SHMT inhibitors

ABSTRACT

The disclosure provides compounds of Formulae (I)-(IX). The disclosed compounds are capable of inhibiting a mammalian SHMT. Compounds of the disclosure have numerous uses, such as for treatment of cancer or autoimmune disorders.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/103,375, filed on Aug. 14, 2018, which is a divisional of U.S.application Ser. No. 15/705,200, filed on Sep. 14, 2017, now U.S. Pat.No. 10,077,273, issued on Sep. 18, 2018, which claims the benefit ofU.S. Provisional Application No. 62/470,131, filed on Mar. 10, 2017, andU.S. Provisional Application No. 62/394,689, filed on Sep. 14, 2016. Theentire teachings of these applications are incorporated herein byreference.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant No. CA163591awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE DISCLOSURE

Serine catabolism is initiated by serine hydroxymethyltransferase (SHMT)activity, catalyzed in the cytosol by SHMT1 and in the mitochondria bySHMT2. SHMTs catalyze a reversible reaction converting serine toglycine, with concurrent methylene-tetrahydrofolate (THF) generation.Increased SHMT enzyme activity has been detected in human breast cancer,colon cancer, and in rat sarcoma.

SHMT functions to generate one-carbon units for cellular folatemetabolism. Inhibition of other aspects of folate metabolism is anestablished mechanism of therapy for a variety of cancers and autoimmunediseases. However, existing anti-folates are characterized bydose-limiting toxicity that limits their effectiveness in cancer therapyand their tolerability in autoimmune disease.

Hypoxia occurs in the tumor environment, and the mitochondrial form ofSHMT, SHMT2, is induced under hypoxic stress. SHMT expression may helptumor cells survive under hypoxic conditions, thus promoting cancerouscell growth, survival and metastasis. Hypoxic cells are generally moreresistant to radiation and chemotherapy treatment, further permittingthe tumor to grow and metastasize. SHMT2 overexpression has beenobserved in various different cancers, including neuroblastoma, bladdercancer, colorectal cancer, kidney cancer, etc.

SUMMARY OF THE DISCLOSURE

There is a need in the art for effective treatments for cancer and otherconditions, such as autoimmune disease. Without being bound by theory,SHMT enzymes are an attractive target and SHMT inhibitors, such asinhibitors of SHMT1 and/or SHMT2, (e.g., selective inhibitors of SHMT2and/or SHMT1) are suitable for a variety of purposes, such as to inhibitSHMT activity in vitro and/or in vivo. Such inhibitors may actadditively or synergistically with other anti-folate compounds, such asto treat cancer and autoimmune disease.

The present disclosure provides compounds, compositions, and methods, asdescribed herein. In certain embodiments, the compounds of thedisclosure are inhibitors of mammalian SHMT activity, such as mammalianSHMT1 and/or mammalian SHMT2 (e.g., human SHMT1 and/or human SHMT2).Compounds of the disclosure may be provided in isolated or substantiallypurified form, including as a substantially pure stereoisomer,enantiomer, diastereomer, atropisomer, and a mixture thereof, and/or maybe provided as compositions, such as pharmaceutical compositions.

In one aspect, the disclosure provides compounds of the disclosures.Compounds of Formula I (including Ia and Ib), Formula II (including IIaand IIb), and Formula III (including IIIa and IIIb), Formula II′(including IIa′ and IIb′), and Formula III′ (including IIIa′ and IIIb′)are exemplary of compounds of the disclosure. The disclosure alsoprovides compounds of any of Formula I-IX (including Ia, Ib, IIa, IIb,IIa′, IIb′ IIa, IIIb, IIIa′ IIIb′, etc., IXa, IXb, and IXc). Compoundsdescribed herein include compounds having any combination of structuraland/or functional features described herein. Similarly contemplated arepharmaceutical compositions comprising any compounds described herein.

In one aspect, compounds of this disclosure are SHMT inhibitors. In someembodiments, the compound of this disclosure is a selective SHMTinhibitor, such as a SHMT1 inhibitor or a SHMT2 inhibitor. In certainembodiments, the compound of this disclosure is a dual SHMT inhibitor,such as an SHMT1 and SHMT2 inhibitor. Any of the compounds of thedisclosure, including compounds described generally or specificallyherein, may be used in any of the in vitro or in vivo methods describedherein, such as in a method of treating cancer or an autoimmunecondition.

In one aspect, the disclosure provides methods for treating a cancer orautoimmune condition, such as a cancer or autoimmune conditionassociated with SHMT activity and/or associated with alterations inmitochondrial folate metabolism. In some embodiments, the cancer orautoimmune condition is associated with mitochondrial dysfunction, suchas alterations in mitochondrial folate metabolism. In some embodiments,the cancer or autoimmune conditions is characterized by the presence ofone or more cells or tissues having mutations in, for example, myc or aprotein important for proper mitochondrial function. Exemplary methodsinclude monotherapy, in which a mammalian patient in need thereof isadministered an SHMT inhibitor that inhibits activity of a mammalianSMHT2 and/or SHMT1. Further exemplary methods include methods in whichan SHMT inhibitor that inhibits activity of a mammalian SHMT2 and/orSHMT1 is administered in a composition and/or as part of a therapeuticregime with one or more additional agents or therapeutic modalities.

In certain embodiments, when multiple agents and/or treatment modalitiesare used as part of the therapeutic method, each such agent may beadministered, independently, at the same or differing times. Similarly,when multiple agents and/or treatment modalities are used as a part ofthe therapeutic method, each such agent may be administered,independently, using the same or differing routes of administrationand/or formulations. All such methods contemplate administration of acompound or a pharmaceutical composition of the disclosure, whetherdescribed generally by function as an SHMT inhibitor, or whetherdescribed using one or more structural and/or functional features, asset forth herein.

In certain embodiments, this disclosure provides a method of treatment(whether as part of a monotherapy or therapeutic regimen) comprisingadministering to a mammalian subject in need thereof, a compound ofFormula (I) (including Ia or Ib), (II) (including IIa or IIb), (III)(including IIIa or IIIb), Formula II′ (including IIa′ or IIb′), andFormula III′ (including IIIa′ or IIIb′), or a pharmaceuticallyacceptable salt thereof, as described herein. Similarly contemplated aremethods in which a pharmaceutical formulation/composition comprising anycompound described herein is used. In certain embodiments, thisdisclosure provides methods comprising administering to a mammaliansubject in need thereof a compound of Formula (I) (including Ia or Ib),(II) (including IIa or IIb), (III) (including IIIa or IIIb), Formula II′(including IIa′ or IIb′), and Formula III′ (including IIIa′ or IIIb′),either as a monotherapy or a combination therapy.

In certain embodiments, this disclosure provides a method comprisingadministration of two or more agents. In certain embodiments, the methodcomprises administration of a selective SHMT inhibitor, such as an SHMT2inhibitor, and a second agent or treatment modality. In certainembodiments, the method comprises administration of a dual SHMTinhibitor and a second agent or treatment modality. In certainembodiments, the second agent is another anti-cancer therapeutic agent,such as a chemotherapeutic agent, an anti-folate, radiation therapy,and/or the then standard of care for the particular cancer or autoimmunecondition being treated.

In certain embodiments, the second agent is a rescue therapy intended tohelp reduce toxicity or otherwise limit side effects. Such rescuetherapy may be used alone with a selective SHMT inhibitor (e.g., a SHMT2inhibitor) or a dual SHMT inhibitor or together with another anti-canceragent, such as a traditional anti-folate. Exemplary rescue therapies andother therapeutic modalities are described herein. In certainembodiments, the second agent is a rescue therapy, such as formate,folinic acid or a derivative thereof (or like compounds, as describedherein), but administration of formate, folinic acid (or like compounds)cooperates to increase the anticancer effects of the SHMT inhibitor. Incertain embodiments, the addition of formate, folinic acid, or similarcompounds improves safety, reduces toxicity or improved the therapeuticindex, such as by maintaining or increasing the anti-cancer effect inthe cancer cells while not doing so or even decreasing any effect inhealthy cells. In other embodiments, the additional of formate decreasestoxicity in healthy cells but also in cancerous cells. When multipleagents or treatment modalities are used as part of a therapeuticregimen, they may be administered at the same or differing times. Forexample, a compound of the disclosure, such as a SHMT inhibitor, may beadministered before, at the same time, or following administration ofanother agent, including a rescue therapy.

In certain embodiments, the cancer is a cancer of a particular tissue,and tumors or cancerous tissue may include cells comprising one or moremutations in, for example, myc or in another gene where the mutation isassociated with mitochondrial dysfunction, such as mutations associatedwith alterations in mitochondrial folate metabolism. It is appreciatedthat tumors and cancerous tissues are typically heterogenous, such thatnot all cells in a tumor will have the same mutational status. Rather,one or more cells of the tumor or cancerous tissue contain such amutation in a mitochondrial enzyme, such as a mitochondrial folateenzyme, or otherwise associated with alterations in mitochondrialmetabolism, such as mitochondrial folate metabolism.

In one aspect, the disclosure provides compounds represented by Formula(I) (including Ia or Ib), (II) (including IIa or IIb), (III) (includingIIIa or IIIb), Formula II′ (including IIa′ or IIb′), and Formula III′(including IIIa′ or IIIb′), or pharmaceutically acceptable salts orstereoisomers thereof, wherein the variables are defined as describedherein. In certain embodiments, such compounds are capable of inhibitingSHMT activity, such as SHMT2 and/or SHMT1 activity (e.g., mammalianSHMT1 and/or mammalian SHMT2, such as human SHMT1 and/or human SHMT2).In certain embodiments, such compounds are selective inhibitors of SHMT2and/or SHMT1. In certain embodiments, such compounds are dual inhibitorsof SHMT1 and SHMT2. Of note, inhibitory activity may be evaluated invitro and/or in vivo.

In certain embodiments, compounds of any of Formula (I) (including Ia orIb), (II) (including IIa or IIb), (III) (including IIIa or IIIb),Formula II′ (including IIa′ or IIb′), and Formula III′ (including IIIa′or IIIb′), or a pharmaceutically acceptable salt thereof, may bedescribed based on any combination of structural and/or functionalfeatures provided herein.

In another aspect, the disclosure provides pharmaceutical compositionscomprising a compound of the disclosure (e.g., a compound of any ofFormula (I) (including Ia or Ib), (II) (including IIa or IIb), (III)(including IIIa or IIIb), Formula II′ (including IIa′ or IIb′), andFormula III′ (including IIIa′ or IIIb′), or a pharmaceuticallyacceptable salt thereof, formulated with one or more pharmaceuticallyacceptable carriers and/or excipients.

In another aspect, the disclosure provides compounds of Formula (I)(including Ia or Ib), (II) (including IIa or IIb), (III) (including IIIaor IIIb), Formula II′ (including IIa′ or IIb′), and Formula III′(including IIIa′ or IIIb′), or stereoisomers thereof, orpharmaceutically acceptable salts thereof, or mixtures of any of theforegoing, or pharmaceutical compositions comprising any one of theforegoing, for use as a medicament.

In another aspect, the disclosure provides numerous methods of usingcompounds and/or compositions of the disclosure alone or in combinationwith other agents or treatment modalities. Compounds and/or compositionsof the disclosure (e.g., a compound of any of Formula I (Ia, or Ib), II(IIa or IIb), or III (IIIa or IIIb), and/or a pharmaceuticallyacceptable salt thereof, and/or a composition comprising any one of theforegoing) may be used in any of the in vitro and/or in vivo methodsdescribed herein. In certain embodiments, the method is a method oftreating cancer. In certain embodiments, the method is a method oftreating an autoimmune condition.

In certain embodiments, this disclosure provides a method for treatingcancer or an autoimmune disorder. The method comprises administering toa mammalian subject in need thereof an effective amount of a compound ofFormula (I) (including Ia or Ib), (II) (including IIa or IIb), (III)(including IIIa or IIIb), Formula II′ (including IIa′ or IIb′), andFormula III′ (including IIIa′ or IIIb′), or a pharmaceuticallyacceptable salt thereof, or a composition comprising any one of theforegoing. In certain such embodiments, the method further comprisesadministering (either before, at the same time, or after) one or moreadditional agents (such as a rescue therapy to reduce toxicity) ortreatment modalities as part of a therapeutic regimen.

In certain embodiments, SHMT inhibitor is an inhibitor of SHMT2 and/orSHMT1.

In certain embodiments, the cancer or autoimmune condition is associatedwith alterations in mitochondrial folate metabolism.

In certain embodiments, the one or more additional agents comprise arescue therapy to reduce toxicity. In other embodiments, the one or moreadditional agents comprise an additional anti-cancer agent. In certainsuch embodiments, the anti-cancer agent is an anti-folate compound.

In certain embodiments, the subject is a human subject.

In certain embodiments, this disclosure provides a method for treatingcancer. In certain embodiments, the cancer comprises a mutation oralteration that affects mitochondrial metabolism, such as a mutation oralteration in a mitochondrial folate pathway gene. By “cancer comprises”it is understood that one or more cells of the tumor or cancerous tissuecontain the mutation or alteration. In certain embodiments, the cancercomprises a mutation or alteration in SHMT2, MTHFD2, MTHFD2L, MTHFD1L,fumarate hydratase (FH), SLC25A32, KEAP1, or NRF2, or the patientotherwise has such a mutation or alteration in non-cancerous tissue.

In certain embodiments, the cancer is selected from pediatric or adultleukemia, lymphoma, solid tumors of the lung, non-small cell lungcancer, mesothelioma, solid tumors of the breast, colon cancer, livercancer, stomach cancer, prostate cancer, pancreatic cancer, ovariancancer, uterus and female genital tract cancer, bladder cancer, head andneck cancer, osteosarcoma, or trophoblastic neoplasms.

In certain embodiments, the cancer is characterized by a Myc mutation.

In certain embodiments, this disclosure provides a method for treatingan autoimmune disorder. In certain such embodiments, the autoimmunedisorder is selected from rheumatoid arthritis, dermatomyositis,psoriasis, lupus, sarcoidosis, Crohn's disease, eczema, or vasculitis.

In certain embodiments the subject in need thereof comprises a mutationor alteration that affects mitochondrial metabolism, such as a mutationor alteration in a mitochondrial folate pathway gene. In certainembodiments, the autoimmune disorder comprises a mutation or alterationthat affects mitochondrial metabolism, such as a mutation or alterationin a mitochondrial folate pathway gene.

In certain embodiments, the rescue therapy is a formate salt or folinicacid. In other embodiments, the rescue therapy comprises: formate,formate salt, formate ester, or leucovorin. In certain embodiments, therescue therapy is a formate, formate salt, formate ester, glycine,leucovorin, or a derivative thereof, or a pharmaceutically acceptablesalt thereof.

In certain embodiments, the disclosure provides SHMT inhibitors for usein treating a condition, such as lymphoma (e.g., diffuse large B-celllymphoma). Exemplary SHMT inhibitors are described herein. In certainembodiments, an SHMT inhibitor is a compound of any of Formulae(I)-(IX), including Formulae (Ia), (Ib), (IIa), (IIb), (IIa′), (IIb′),(IIIa), (IIIb), (IIIa′), (IIIb′), etc., (IXa), (IXb), (IXc).

In certain embodiments, the disclosure provides a method for treatinglymphoma, such as T-cell lymphoma, B-cell lymphoma, or NK-cell lymphoma,comprising administering to a mammalian subject in need thereof aneffective amount of an SHMT inhibitor. In certain such embodiments, thelymphoma is a B-cell lymphoma. In some embodiments, the lymphoma is adiffuse large B-cell lymphoma. In certain embodiments, the disclosureprovides a method for inhibiting proliferation or survival of B-celllymphoma cells, comprising contacting such cells with an effectiveamount of an SHMT inhibitor and/or a composition comprising such an SHMTinhibitor, e.g., a compound an/or composition of this disclosure. Incertain embodiments, the method is an in vivo method. In certainembodiments, the method is an in vitro method. In certain embodiments,the disclosure provides methods for inhibiting growth or promotinggrowth arrest of B-cell lymphoma cells, comprising contacting such cellswith an effective amount of an SHMT inhibitor. In certain embodiments,the method is an in vivo method. In certain embodiments, the method isan in vitro method.

In certain embodiments, the method comprises administering formate or aderivative thereof (as described herein). Although formate alone rescuesthe effects of an SHMT inhibitor in many cell types, surprisingly, theformate rescue effects were not observed in B-cell lymphomas, such asdiffuse large B-cell lymphoma, where a combination of formate andglycine (such as supraphysiologic glycine) is required to so rescue.Rather, although formate (or derivatives thereof, as described herein)is traditionally classified as a rescue therapy, it is suitable for usein combination with an SHMT inhibitor in B-cell lymphoma where itactually further increases the inhibitory effect on the cancerous cellsof the SHMT inhibitor. Accordingly, the disclosure provides improvedmethods of treating B-cell lymphoma, such as diffuse large B-celllymphoma, using a combination of an SHMT inhibitor (such as a SHMT1inhibitor, a SHMT2 inhibitor, or a dual SHMT inhibitor) and/or a rescuetherapy (e.g., formate, folinic acid, etc.), but wherein the combinationactually has increased inhibitory activity against the B-cell lymphoma.In some embodiments, the combination has an improved therapeutic indexagainst cancer, such as B-cell lymphoma.

In certain embodiments, the method further comprises administering (orcontacting cells with) at least one additional agent. In certainembodiments, at least one additional agent is a rescue therapy to reducetoxicity. In certain such embodiments, the SHMT inhibitor is a compoundof the disclosure (a compound generically or specifically disclosedherein, such as a compound of any of Formulae (I)-(IX), or apharmaceutical composition comprising a compound of any of Formulae(I)-(IX). In certain embodiments, the SHMT inhibitor is formulated withone or more pharmaceutically acceptable carriers and/or excipients. Incertain embodiments, the SHMT inhibitor is a compound of any of Formula(I) (including Ia or Ib), (II) (including IIa or IIb), (III) (includingIIIa or IIIb), Formula II′ (including IIa′ or IIb′), and Formula III′(including IIIa′ and IIIb′). In certain embodiments, the SHMT inhibitoris a compound of any of Formulae (IV)-(IX).

In certain embodiments, the rescue therapy is a formate salt or folinicacid, or a derivative thereof. In other embodiments, the rescue therapycomprises: formate, formate salt, formate ester, or leucovorin, or aderivative thereof. In certain embodiments, the rescue therapy is aformate, formate salt, formate ester, glycine, leucovorin, or aderivative thereof, or a pharmaceutically acceptable salt thereof. Incertain embodiments, the additional agent is glycine or formate, aderivative thereof, or a pharmaceutically acceptable salt thereof. Insome embodiments, the additional agent is glycine, a derivative thereof,or a pharmaceutically acceptable salt thereof. In other embodiments, theadditional agent is formate, a derivative thereof, or a pharmaceuticallyacceptable salt thereof. In yet other embodiments, the rescue theory isa combination of glycine and formate, or derivatives thereof, orpharmaceutically acceptable salts thereof. In some embodiments, theformate and/or glycine rescue is sufficient to restore glycine levels inthe normal body (e.g., a healthy cell or tissue) but not in a tumor suchas lymphoma.

In certain embodiments, the method further comprises administering (orcontacting cells with) a glycine uptake inhibitor, such as RG1678(Bitopertin), Org 24598, Org 25935, ALX-5407, sarcosine, Org25543,N-arachidonylglycine, amoxapine, or ethanol. In certain suchembodiments, the glycine uptake inhibitor is RG1678 (Bitopertin). Incertain embodiments, the glycine uptake inhibitor is a glycinetransporter (e.g., glycine transporter type-1 (GlyT-1), glycinetransporter type-2 (GlyT-2)) inhibitor. In certain embodiments, theglycine uptake inhibitor is a GlyT-1 inhibitor. As used herein, “glycineuptake inhibitor” refers to agents that inhibit the uptake or reuptakeof glycine, for example, by inhibiting a glycine transporter.

In certain embodiments, the SHMT inhibitor is an inhibitor of SHMT2and/or SHMT1.

In certain embodiments, the B-cell lymphoma is a Hodgkin's lymphoma(HL), such as a classical HL or nodular lymphocyte-predominant HL (e.g.,nodular sclerosis HL, mixed cellularity HL, lymphocyte-rich HL, orlymphocyte-depleted HL).

In certain embodiments, the B-cell lymphoma is a non-Hodgkin's lymphoma(NHL), such as a diffuse large B-cell lymphoma (e.g., a primarymediastinal large B-cell lymphoma, T-cell/histiocyte-rich large B-celllymphoma, or intravascular large B-cell lymphoma).

In certain embodiments, the B-cell lymphoma is a diffuse large B-celllymphoma (DLBC lymphoma or DLBCL), Burkitt lymphoma, follicularlymphoma, marginal zone B-cell lymphoma, extranodal marginal zonelymphoma, mucosa-associated lymphatic tissue lymphoma, small lymphocyticlymphoma, or mantle cell lymphoma. In some embodiments, the B-celllymphoma is a diffuse large B-cell lymphoma. In other embodiments, theB-cell lymphoma is Burkitt lymphoma.

In certain embodiments, the subject is a human subject (e.g., a humancancer patient).

The disclosure contemplates combinations of any of the aspects and/orembodiments described herein. Compounds of the disclosure may bedescribed based on any suitable combination (e.g., as valence andstability permit) of structural and/or functional properties providedherein. For example, any of the compounds described herein, such as anyof the SHMT inhibitors (e.g., compounds that inhibit activity ofmammalian SHMT2 and/or SHMT1) described herein, may be used in thetreatment of any of the conditions described herein, such as byadministering an effective amount to a subject in need thereof.Similarly, any of the compounds described herein may be provided ascompositions, such as pharmaceutical compositions, and any suchpharmaceutical compositions may be used in the treatment of any of theconditions described herein. Similarly, compounds or compositions of thedisclosure may be used in vivo or in vitro, such as in any of themethods, described herein.

DETAILED DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A illustrates that deuterated serine (2,3,3-²H₃ serine,(D3-serine)) isotopic labeling into deoxythymidine phosphates revealsmitochondrial versus cytosolic compartmentalization of serinemetabolism.

FIG. 1B illustrates that serine synthesis and catabolism occur in aninter-compartmental cycle mediated by cytosolic and mitochondrial SHMTactivity. Key enzymes mediating these transformations are highlighted incapital letters.

FIG. 2 shows inhibition of human SHMT enzyme activity (using calculatedpercent inhibition) by potent pyrazolopyran compound described in thisdisclosure against human SHMT1 (FIG. 2A) and SHMT 2 (FIG. 2B) in an invitro assay.

FIG. 3 shows cancer cell growth inhibition of potent pyrazolopyrancompounds described in this disclosure. Compounds of this disclosuredemonstrated different inhibition effects against wildtype (FIG. 3A) andSHMT2 knock-out (FIG. 3B) human colon carcinoma HCT-116 cells.

FIG. 4 shows B-cell lymphoma lines are sensitive to SHMT inhibitors andnot rescued by formate. The diffuse large B-cell lymphoma cell line,Su-DHL-2, was sensitive to the SHMT inhibitor, (+)-HK-16, and the growthinhibitory effect was not rescuable by formate. In contrast, the JurkatT cell line was sensitive to the same SHMT inhibitor, but the growtharrest was rescuable by treatment with formate. Compare FIG. 4A (Jurkatcells) with FIG. 4B (Su-DHL-2 cells).

FIG. 5 shows SHMT inhibitor (SHMTi) growth inhibitory activity can berescued in Su-DHL-2 cells (DLBCL B cell lymphoma cells) with acombination of formate and glycine. Su-DHL-2 cells were cultured with 5μM of an SHMTi. Growth was rescued by combination with 1 mM sodiumformate and 100 mg/L glycine (10× standard RPMI concentration).

FIG. 6 shows a combination of supplemental formate and glycine isrequired to rescue B-cell lymphoma lines from SHMTi inhibition. Diffuselarge B-cell lymphoma (DLBCL) lines (Su-DHL-2, Su-DHL-4, Farage andSu-DHL-6) were cultured in RPMI with 5 μM of an SHMT inhibitor (SHMTi).Growth was rescued by addition of formate (1 mM sodium formate) ANDglycine (100 mg/L). In a representative AML cell line, REH, cell growthwas rescued by formate alone.

FIG. 7 shows comparison studies of glycine import using ADP labeling invarious cell lines to demonstrate impaired glycine uptake in B-cellmalignancies. FIG. 7A provides a schematic showing contribution ofglycine to de novo synthesis of adenosine ring. FIG. 7B shows theresults of isotopic labeling (¹³C) of ADP extracted from variousadherent solid tumor derived cancer cell lines incubated with 1,2-¹³Cglycine for 48 hours as determined by mass spectrometry. In all suchsolid tumor cell lines tested, a substantial fraction of ADP is M+2labeled, indicating incorporation of ¹³C glycine from the media. FIG. 7Cshows the results of ADP ¹³C labeling after 48 hour incubation with1,2-¹³C glycine in B-cell cancer lines. Results from cell lines fromboth Burkitt lymphoma and DLBCL reveal nearly no incorporation ofexogenous glycine (i.e., imported from outside the cell) into ADP(absence of M+2 labeling in 7C).

FIG. 8 shows SHMT is required for tumor formation in vivo. FIG. 8A showsgrowth growth of subcutaneous tumors from HCT-116 WT and ΔSHMT2 cellsimplanted in opposite flanks of nude mice (mean±SEM, n=10, * p<0.05,paired t-test). FIG. 8B shows intratumor abundance of AICAR and serinefrom xenografted tumors (n=9, *** p<0.001, paired t-test). FIG. 8C showsgrowth of HCT-116 WT and ΔSHMT1/2 double deletion cells in standard DMEMwith and without supplemental 1 mM sodium formate (n≥4). FIG. 8D showstumor growth of subcutaneous tumors from HCT-116 ΔSHMT2 and ΔSHMT1/2cells implanted in opposite flanks of nude mice (mean±SEM, n=10).

FIG. 9A shows enzymatic inhibition curves for human SHMT1 and SHMT2 withenantiomerically resolved fractions (enantiomer A and enantiomer B) ofCompound HK-X1.

FIG. 9B shows a magnified view of Compound HK-X1 in complex with humanSHMT2 as solved in a 2.5 Å resolution x-ray crystal structure. Theelectron density of the compound is shown as the 2F_(o)-F_(c) mapcontoured at 0.5σ and generated with Compound HK-X1 omitted.

FIG. 9C shows an overlay of the SHMT2/Compound HK-X1 structure from FIG.9B with the structure of 5-formyl-THF-triglutamate in complex withrabbit SHMT1.

FIG. 9D shows growth of HCT-116 WT±1 mM formate and ΔSHMT1 and ΔSHMT2cell lines in the presence of increasing concentrations of CompoundHK-X2 (n≥3). Compound HK-X1 and compounds of Fomulae (I)-(III) of thisdisclosure have shown similar inhibitory effects against HCT-116 WT andΔSHMT1 and ΔSHMT2 cell lines (data not shown).

FIG. 9E shows growth of human pancreatic cell line 8988T with indicatedconcentrations of SHMT inhibitor Compound (+)-HK-16. Compound HK-X1,HK-X2 and compounds of Formulae (I)-(III) of this disclosure have shownsimilar inhibitory effects against HCT-116 WT and ΔSHMT1 and ΔSHMT2 celllines (data not shown).

FIG. 10 shows Compound (+)-HK-X2 inhibits SHMT1 and SHMT2 in HCT-116cells. FIG. 10A is a schematic illustration of isotope labeling fromU-13C-serine into downstream metabolites. Heavy (13C) atoms arerepresented by filled in circles. FIG. 10B shows (top) a fraction oforiginal serine remaining in media after 24 hours incubation U-¹³Cserine Compound HK-X2 and (bottom) M+1 13C-labeling fraction of mediaserine after 24 hours (mean±SD, n=3). FIG. 10C shows M+2 ¹³C-labelingfraction of intracellular ADP and glutathione after 24 h ¹³C-serineco-incubation with DMSO or 5 μM (+)-HK-X2 (mean±SD, n=3). FIG. 10D showsnormalized (to DMSO WT HCT-116 cells) levels of purine biosyntheticpathway intermediates after 24 hours incubation±Compound HK-X2 (mean±SD,n=3). FIG. 10E shows growth of HCT-116 WT cells over 48 hours culturedwith varying concentrations of Compound HK-X2±1 mM sodium formate(mean±SD, n=3). FIG. 10F shows total metabolite abundances in HCT-116cells treated with DMSO versus Compound (+)-HK-X2 for 48 hours. FIG. 10Gshows metabolite abundance in HCT-116 cells treated with DMSO orCompound HK-X2 in the presence of 1 mM sodium formate. Metabolites whoseabundances differ by more than 4-fold between conditions are highlightedin red (mean, n=3).

FIG. 11 shows SHMT inhibitors are particularly active against B-cellmalignancies. FIG. 11A shows ranked IC50 of Compound (+)-HK-X1 forgrowth inhibition of 298 human cancer cell lines. Lines of B-cell originare highlighted in red and are enriched among the more sensitive cells(IC50<4 μM). FIG. 11B shows IC50 of (+)-HK-X2, with and without 1 mMformate, for growth inhibition of select hematological cell lines. FIG.11C shows representative flow cytometry histograms of B-cell lineSu-DHL-4 treated with (+)-HK-X2 (5 μM) and formate (1 mM). Etoposide wasused as a positive control. Cells were stained with propidium iodide andFITC conjugated Annexin V. 10,000 events shown. FIG. 11D shows fractionof Su-DHL-4 and Jurkat cells that are apoptotic (Annexin V+, PI−) 24hours after indicated treatment (mean±SD, n≥3).

FIG. 12 shows sensitization of malignancies of B-cell origin toco-treatment with Compound HK-X2 and formate due to poor uptake ofextracellular glycine. FIG. 12A shows the steady-state labeling fractionof intracellular metabolites synthesized from glycine in select cancercell lines cultured in RPMI containing U-¹³C-glycine (mean±SD, n=3).FIG. 12B shows intracellular ¹³C-glycine assimilation kinetics in Jurkat(fast glycine uptake) and Su-DHL4 (slow glycine uptake) (mean±SD, n=3).FIG. 12C shows doubling times of cells cultured in RPMI with and withoutglycine and/or formate and Compound HK-X2 at doses of comparable potencyfor Jurkat (2.5 μM=IC₈₀) and SU-DHL4 (5 μM=IC₇₀). FIG. 12D showsrepresentative flow cytometry histograms of Su-DHL4 cells treated withindicated combinations of Compound HK-X2 and formate. Cells were stainedwith propidium iodide and FITC conjugated Annexin V. 10,000 eventsshown. FIG. 12E shows fraction of Su-DHL4 cells that are apoptotic(Annexin V+, PI−) 24 hours after indicated treatment (mean±SD, n=3).FIG. 12F shows sensitivity of Su-DHL4 cells to Compound HK-X2 isdependent upon media glycine concentration. Cells were cultured ineither normal RPMI (10 mg/L glycine) or RPMI containing 10× glycine (100mg/L) and Compound HK-X2 for 48 hours, and growth was measured byresazurin assay (mean±SD, n=3). FIG. 12G shows cell growth (normalizedto DMSO) of diffuse large B-cell lymphoma and other hematopoietic cancerlines with 2.5 μM Compound HK-X2, in RPMI with or without 1 mM formateand 10× physiological glycine (100 mg/L). All conditions included atleast normal media glycine (10 mg/L) (mean±SD, n=3).

FIG. 13A shows that SLC38A2 loss is correlated with sensitivity to SHMTinhibitor+formate. Spearman rank correlation of mRNA expression (CCLEdatabase) of known amino acid transporters and sensitivity (IC50) ofselect cancer cell lines (of HEME origin) to inhibition by an SHMTinhibitor with 1 mM sodium formate. Positive correlation is between lowexpression and low IC50. Loss of this amino acid transporter ishypothesized to impair glycine transport and thus sensitize cells toSHMT inhibition in combination with formate.

FIG. 13B shows that SLC38A2 expression is weakly correlated withsensitivity to SHMT inhibitor without formate. Spearman rank correlationof mRNA expression (CCLE database) of known amino acid transporters andsensitivity (IC50) of select cancer cell lines (of heme origin) toinhibition by SHMT inhibitor. Positive correlation is between lowexpression and low IC50.

FIG. 14 shows that glycine made by SHMT is required for B-lymphoma cellline growth. FIG. 14A shows normalized total ion counts of nucleotidetriphosphates in Jurkat ALL cells and Su-DHL-4 DLBCL cells after 72 hour(h) treatment with (+)-HK-X2 (5 Co-culture with 1 mM formate restoresnucleotide levels selectively in Jurkat cells (mean±SD, n=3-6). FIG. 14Bshows normalized glutathione levels from Jurkat and Su-DHL-4 cellstreated as in FIG. 14A (mean±SD, n=3-6). FIG. 14C shows growth ofSu-DHL-4 cells treated with (+)-HK-X2 and hypoxanthine (100 μM) orthymidine (16 μM) (mean±SD, n=3). FIG. 14D shows intracellularU-¹³C-glycine assimilation kinetics in Jurkat and Su-DHL-4. Note the lowuptake in Su-DHL-4 cells (gly=glycine, GSH=glutathione) (mean±SD, n=3).FIG. 14E shows the steady-state labeling fraction of intracellularmetabolites synthesized from glycine in cancer cell lines cultured inRPMI containing U-¹³C-glycine (mean±SD, n=3). FIG. 14F shows cell growth(or death) as measured by log₂ fold change in cell number over 48 h inSu-DHL-4 cells cultured in RPMI with and without glycine (10 mg/L),formate (1 mM), the glycine reuptake transporter 1 inhibitor RG1678 (300nM) and/or (+)-HK-X2 (5 μM) (mean±SD, n=3). FIG. 14G shows a schematicillustrating the proposed glycine vulnerability in B-cells. The SHMTreaction makes two products, 5,10-methyleneTHF and glycine. When SHMT isinhibited, exogenous formate can be incorporated into the 1C cyclewhereas in B-cells poor glycine uptake limits the ability ofextracellular glycine to rescue. Strategies that further stress glycineavailability augment the efficacy of SHMT inhibition.

DETAILED DESCRIPTION OF THE DISCLOSURE A. Overview

Serine hydroxymethyltransferase (SHMT) is an enzyme which plays animportant role in cellular one-carbon pathways by catalyzing thereversible conversions of L-serine to glycine. In addition, SHMTcatalyzes the conversion of tetrahydrofolate to5,10-methylenetetrahydrofolate (e.g., SHMTs catalyze a reversiblereaction converting serine to glycine, with concurrentmethylene-tetrahydrofolate (meTHF) generation). SHMT enzymatic activityprovides the largest part of the one-carbon units available to the cell.In mammals, such as humans, there are two isoforms of SHMT: SHMT1 in thecytosol and SHMT2 in the mitochondria.

The mammalian enzyme is a tetramer of four identical subunits ofapproximately 50,000 Daltons each. The intact holoenzyme in vivo has amolecular weight of approximately 200,000 Daltons and incorporates fourmolecules of vitamin B₆ as a coenzyme.

Human beings have an absolute dietary requirement for folic acid, theessential cofactor for one-carbon metabolism, and adequate levels arenecessary for both normal embryological development and adult tissuefunction. It has long been recognized that proliferative tissues areparticularly dependent upon one-carbon metabolism, and this led to thedevelopment of the first effective chemotherapy, the anti-folates. Theseagents, including methotrexate, and the more recently approvedpemetrexed, are routinely used in the treatment of a variety of cancers,including non-small cell lung cancer (NSCLC), osteosarcoma,mesothelioma, breast cancer, and multiple hematological malignancies.However, the utility of these medications is limited by toxicitiesarising from antifolate activity in normal tissue, which include anemia,neutropenia, diarrhea, and alopecia.

These toxicities often necessitate administration of leucovorin as arescue therapy, although new evidence suggests that folate rescuetherapy combined with traditional anti-folates may contribute to drugresistance. These traditional antifolate therapies are believed tofunction mainly through inhibition of the cytosolic folate enzymesdihydrofolate reductase (DHFR) and thymidylate synthetase (TS),resulting in impaired DNA synthesis and impaired cellular replication.This model is consistent with the toxicity profiles observed in patientswho receive antifolate therapies. In contrast, modulation of serine fluxand/or folate metabolism via inhibition of SHMT may provide the benefitsof traditional antifolate therapies without the deficiencies of anapproach based on inhibiting DHFR and/or TS (e.g., traditionalantifolates; alternative anti-folates). Moreover, rescue therapy canstill be used, in combination, to help further decrease or managetoxicity.

The present disclosure provides compounds, compositions, and methodssuitable for treating cancer and autoimmune conditions, including insubjects having mutations or alterations affecting native mitochondrialfunction or a native mitochondrial folate pathway. Without being boundby theory, even in subjects without known alterations in mitochondrialmetabolism, SHMT inhibitors are suitable for altering folate metabolism,and thus, depriving cells of the energy necessary to fuel pathologicalgrowth and activity. Accordingly, these agents that modulate folatemetabolism in the mitochondria and, potentially, in the cytoplasm, havesignificant utility in modulating cell behavior in numerous contextsincluding cancer and autoimmune conditions.

Folate metabolism occurs as a cycle between two interconnected pathways:one in the cytosol, which directly contributes one-carbon (1C) units tocellular biosynthetic processes, and one in the mitochondria. Thepathways are connected by the metabolites serine, glycine, and formate.Since most studies on folate metabolism initially revolved around theactions of antifolate therapy and the cellular pathophysiology offolate/vitamin B12 deficiency, both of which were believed to involvecytosolic enzymes, the role of the mitochondrial pathway in regulating1C metabolism was underappreciated until much later. Since itselucidation, however, it has been shown that folate metabolism in themitochondrial compartment is central to eukaryotic 1C metabolism and isan original source for the majority of 1C units in the tissue systemsstudied to date.

Cancer growth and proliferation are supported by disease-specificmetabolic processes. In cancer, these include enhanced glucose uptake,aerobic glycolysis (the Warburg effect), and folate-dependent one-carbon(1C) flux. The predominatnt source of 1C units in cancer cells is theamino acid serine. The enzyme SHMT catalyzes the conversion of serineand tetrahydrofolate (THF) into clycine and 5, 10-methylene-THF (meTHF).Increases in the synthesis and consumption of serine and glycine havebeen identified in many transformed cells and cancers. In cancer,mitochondrial serine hydroxymethyl transferase (SHMT2) and the immediatedownstream enzyme, mitochondrial methylene tetrahydrofolatedehydrogenase (MTHFD2), which forms the core of the mitochondrialpathway, are highly expressed in multiple cancer types and sit in thecenter of the cancer-induced 1C metabolic network, linking higher serinesynthesis by phosphoglycerate dehydrogenase (PHGDH) to the mitochondrialproduction and subsequent cytosolic utilization of 1C units fornucleotide synthesis. Comparative oncogenomics identifies PSMB4 andSHMT2 as potential cancer driver genes. Metabolic enzyme expressionhighlights a key role for MTHFD2 and the mitochondrial folate pathway incancer. In contrast, expression of SHMT2 in normal adult tissue wasfound to be consistently low, even in most rapidly proliferating tissuesexamined. In a separate study, high SHMT2 expression correlated withlower overall survival in lung cancer patients, and overexpression ofthis pathway was associated with a poor prognosis in breast cancer.

High expression of SHMT2 and MTHFD2 is linked to rapid catabolism ofserine into glycine and folate associated 1C units. In contrast,cytosolic 1C metabolism is not consistently upregulated in cancer. Thisreflects most cancer cells defaulting to mitochondrial serine catabolismby SHMT2, with the cytosolic isozyme SHMT1 playing a minimal role (andactually not consuming 1C units to synthesize serine), as demonstratedby isotope tracer studies with 2H-serine tracer, which can distinguish1C units generated by mitochondrial versus cytosolic serine catabolism.Without being bound by theory, high expression of core mitochondrialfolate enzymes in cancer is consistent with the role of serine flux(which provides these 1C units) through the mitochondria in cancer. Apotential outcome of higher serine flux through this compartment isincreased export of formate from the mitochondria, effectivelyaugmenting the cytosolic 1C pool.

While the mitochondrial pathway usually supplies all of the 1C units inrapidly proliferating cells, it is not essential in nutrient repleteconditions, as evidenced by the viability of SHMT2 and MTHFD2 deletioncell lines. In such deletion cells, SHMT1 reverses direction andproduces 1C units required for purine and thymidine synthesis. In theabsence of exogenous glycine, however, this flux is not sufficient tomeet glycine demand and these and all mitochondrial folate mutant celllines are glycine auxotrophs.

1C metabolism is targeted therapeutically by folate analogues, includingthe common clinical agents pemetrexed and methotrexate. These drugsmimic folate and broadly block folate metabolism. Another commonchemotherapeutic drug, 5-fluorouracil, specifically targets 1Cutilization to make thymidine (in addition to thymidine incorporationinto DNA). No existing antifolates are thought to specifically targetthe production of 1C units from serine, the primary source of 1C intumors. Efforts to block serine synthesis through inhibition of PHGDHhave been largely unsuccessful as tumor cells avidly uptakeenvironmental serine, bypassing the requirement for PHGDH.

In addition, and without being bound by theory, roles for SHMT2 incancer may also include a non-biosynthetic role of mitochondrial folatemetabolism in cancer as a redox defense through generation of NADPHwithin the mitochondria (e.g., such as a defense to hypoxic conditions).In addition, and without being bound by theory, the role for SHMT2 incancer may also include 5,10-methyleneTHF dependent translation ofmitochondrial proteins and maintenance of mitochondrial electrontransport function, oxidative phosphorylation and organelle specificmetabolic activity. In addition, and without being bound by theory, therole for SHMT2 in cancer may also include a non-1C metabolismbiosynthetic contribution in the form of glycine generation bothspecifically localized to within the mitochondria and in the cytosol.Glycine synthesis can support mitochondrial health and function as wellas the redox state of the entire cell.

Modulation of folate metabolism is suitable for therapeuticintervention, such as in cancer. The present disclosure providesinhibitors of SHMT2 and/or SHMT1. Such inhibitors represent analternative to traditional anti-folates or anti-folates that directlyinhibit DHFR or TS for modulating folate metabolism in cells in vitroand/or in vivo. Thus, in certain embodiments, the disclosure providescompounds, compositions and methods for modulation of serine flux and/orfolate metabolism using inhibitors, such as selective inhibitors, ofmammalian SHMT enzymes. In certain embodiments, the disclosure providescompounds, compositions, and methods to modulate (e.g., inhibit) serineflux and/or the mitochondrial folate pathway by inhibiting SHMT2 (e.g.,using inhibitors of SHMT2; providing compounds capable of inhibitingSHMT2). Such inhibitors may optionally also inhibit SHMT1. Similarly, incertain embodiments, the disclosure provides compounds, compositions,and methods to modulate (e.g., inhibit) generation of NADPH byinhibiting SHMT2 (e.g., using inhibitors of SHMT2; providing compoundscapable of inhibiting SHMT2). Such inhibitors may optionally alsoinhibit SHMT1.

In certain embodiments, the disclosure provides compounds, compositions,and methods for modulating (e.g., inhibiting) glycine generation in themitochondria and/or cytosol in cells. Accordingly, the disclosureprovides, in certain embodiments, compounds and/or compositions capableof inhibiting a mammalian SHMT enzyme (e.g., SHMT2 and/or SHMT1), aswell as methods for using such compounds. In certain embodiments, suchinhibitors of a mammalian SHMT2 and/or SHMT1 are selective inhibitorsfor SHMT enzymes (e.g., the compounds show selectivity for SHMT enzymesover DHFR and/or TS and/or MTHFD2). In certain embodiments, suitableinhibitors of mammalian SHMT2 and/or SHMT1 do not substantially inhibitthe activity of DHFR and/or TS and/or MTHFD2.

In one aspect, the present disclosure provides SHMT inhibitors,including selective and dual SHMT (SHMT1 and SHMT2) inhibitors. It hasbeen shown that dual SHMT1/2 knockout prevents xenograft formationaccording to Examples of this disclosure. Dual SHMT inhibition blockscell growth in a formate rescuable fashion and phenocopies geneticknockout. However, in some hematological cell lines, for example B-celllymphoma lines such as diffuse large-cell B-cell lymphomas (DLBCL),formate alone did not rescue, but rather synergized with an SHMTinhibitor (such as an SHMT inhibitor described herein) as shown in theExamples of this disclosure. This unexpected outcome reflects theinability of these cells to uptake glycine, a required product of theSHMT reaction. Without being bound by theory, defective glycine uptakerenders DLBCL cells (as well as other cancers in which formate fails torescue following treatment with an SHMT inhibitor) uniquely sensitive toSHMT inhibition (alone or in combination with formate or a formatederivative).

These SHMT inhibitors are useful in numerous in vitro and in vivoapplications, as described herein, including in the treatment of cancerand other hyperproliferative conditions, as well as in the treatment ofautoimmune disorders, particularly those caused or exacerbated byproliferation or increased metabolic activity of immune cells. Incertain embodiments, SHMT inhibitors are useful in cancers or othercontexts that are associated with alterations in mitochondrialmetabolism, such as mitochondrial folate metabolism (e.g., cancers thatcontain alterations in genes associated with mitochondrial metabolism).Without being bound by theory, such cancers seem to be particularlysensitized to SHMT inhibitors.

B. Definitions

Unless otherwise defined herein, scientific and technical terms used inthis disclosure shall have the meanings that are commonly understood bythose of ordinary skill in the art. Generally, nomenclature used inconnection with, and techniques of, chemistry, cell and tissue culture,molecular biology, cell and cancer biology, immunology, andpharmacology, described herein, are those well known and commonly usedin the art.

Chemistry terms used herein are used according to conventional usage inthe art, for example as exemplified by “The McGraw-Hill Dictionary ofChemical Terms”, Parker S., Ed., McGraw-Hill, San Francisco, Calif.(1985).

All of the above, and any other publications, patents and publishedpatent applications referred to in this disclosure are specificallyincorporated by reference herein. In case of conflict, the presentdisclosure, including its specific definitions, will control.

It is to be understood that the present disclosure is not limited toparticular embodiments described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

It is appreciated that certain features of the disclosure, which are,for clarity, described in the context of separate embodiments, may alsobe provided in combination in a single embodiment. Conversely, variousfeatures of the disclosure, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present disclosure and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterized, and tested for biological activity). In addition, allsubcombinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentdisclosure and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

Any formula depicted herein is intended to represent a compound of thatstructural formula as well as certain variations or forms. For example,a formula given herein is intended to include a racemic form, or one ormore enantiomeric, diastereomeric, or geometric isomers, or tautomericforms, or a mixture thereof. Additionally, any formula given herein isintended to refer also to a solvate, such as a hydrate, solvate, orpolymorph of such a compound, or a mixture thereof. Any formula givenherein is intended to refer to amorphous and/or crystalline physicalforms of the compound. The compounds described herein may beanalytically pure, or a mixture in which the compound comprises at least50%, at least 70%, at least 80%, at least 90%, at least 95%, or at least98% by weight of the mixture.

In addition, where features or aspects of the embodiments of thisdisclosure are described in terms of Markush groups, those skilled inthe art will recognize that embodiments described herein is also therebydescribed in terms of any individual member or subgroup of members ofthe Markush group. For example, if X is described as selected from thegroup consisting of bromine, chlorine, and iodine, claims for X beingbromine and claims for X being bromine and chlorine are fully described.

The term “herein” refers to the entire disclosure.

As used herein, the terms “including,” “containing,” “comprises,” and“comprising” are used in their open, non-limiting sense.

The term “alkoxy” refers to an oxygen atom having an alkyl groupattached thereto. Representative alkoxy groups include methoxy, ethoxy,propoxy, tert-butoxy and the like. In some embodiments, a straight chainor branched chain alkoxy has 30 or fewer carbon atoms, and preferably 20or fewer, such as C₁-C₁₀ alkoxy, C₁-C₈ alkoxy, or C₁-C₆ alkoxy.

The term “alkenyl” refers to an aliphatic group containing at least onedouble bond and is intended to include both “unsubstituted alkenyls” and“substituted alkenyls”, the latter of which refers to alkenyl moietieshaving substituents replacing a hydrogen on one or more carbons of thealkenyl group. Such substituents may occur on one or more carbons thatare included or not included in one or more double bonds. Moreover, suchsubstituents include all those contemplated for alkyl groups, asdiscussed below, except where stability is prohibitive. For example,substitution of alkenyl groups by one or more alkyl, carbocyclyl, aryl,heterocyclyl, or heteroaryl groups is contemplated. In some embodiments,a straight chain or branched chain alkenyl has 30 or fewer carbon atoms,and preferably 20 or fewer, such as C₂-C₁₀ alkenyl or C₂-C₈ alkenyl.

The term “alkynyl” refers to an aliphatic group containing at least onetriple bond and is intended to include both “unsubstituted alkynyls” and“substituted alkynyls,” the latter of which refers to alkynyl moietieshaving substituents replacing one or more hydrogens on one or morecarbons of the alkynyl group. Such substituents may occur on one or morecarbons that are included or not included in one or more triple bonds.Moreover, such substituents include all those contemplated for alkylgroups, as discussed below, except where stability is prohibitive. Forexample, substitution of alkynyl groups by one or more alkyl,carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.In some embodiments, a straight chain or branched chain alkynyl has 30or fewer carbon atoms, and preferably 20 or fewer, such as C₂-C₁₀alkynyl or C₂-C₈ alkynyl.

The term “alkyl” refers to a saturated aliphatic groups, includingstraight-chain alkyl groups, and branched-chain alkyl groups. Inpreferred embodiments, a straight chain or branched chain alkyl has 30or fewer carbon atoms in its backbone (e.g., C₁-C₃₀ for straight chains,C₃-C₃₀ for branched chains), and more preferably 20 or fewer. In certainembodiments, alkyl groups are lower alkyl groups, e.g., methyl, ethyl,n-propyl, i-propyl, n-butyl and n-pentyl. In certain embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains). In preferred embodiments, the chain has ten or fewer carbon(C₁-C₁₀) atoms in its backbone. In some embodiments, the chain has eightor fewer carbon (C₁-C₈) atoms. In other embodiments, the chain has sixor fewer carbon (C₁-C₆) atoms in its backbone. A C₁-C₆ straight chainedor branched alkyl group is also referred to as a “lower alkyl” group.Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thedisclosure, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone.

Such substituents can include, but not limited to, for example, ahalogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl,a formyl, or an acyl), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, an alkylthio, an acyloxy, aphosphoryl, a phosphate, a phosphonate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aryl or heteroaryl moiety. It will beunderstood by those skilled in the art that the moieties substituted onthe hydrocarbon chain can themselves be substituted, if appropriate. Forinstance, the substituents of a substituted alkyl may includesubstituted and unsubstituted forms of hydroxyl, halo, amino, azido,imino, amido, phosphoryl (including phosphonate and phosphinate),sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), andsilyl groups, as well as ethers, alkylthios, carbonyls (includingketones, aldehydes, carboxylates, and esters), —CF₃, —CN and the like.Exemplary substituted alkyls are described below. Cycloalkyls can befurther substituted with alkyls, alkenyls, alkoxys, alkylthios,aminoalkyls, carbonyl-substituted alkyls, —CF₃, —CN, and the like.

The term “arylkyl”, as used herein, refers to an alkyl group substitutedwith one or more aryl groups.

The term “aryl”, as used herein, includes substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. Aryl groups include phenyl, phenol, aniline, and thelike.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings wherein at least one of the rings is aromatic, e.g., the othercyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls. Aryl groups include benzene,naphthalene, phenanthrene, phenol, aniline, and the like.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

wherein each R³⁰ independently represents a hydrogen or a hydrocarbylgroup, or two R³⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The term “aminoalkyl” refers to an alkyl group substituted with an aminogroup.

The term “amide” refers to a group:

wherein each R³⁰ independently represent a hydrogen or hydrocarbylgroup, or two R³⁰ are taken together with the N atom to which they areattached complete a heterocycle having from 4 to 8 atoms in the ringstructure.

The terms “nitrile” or “cyano,” as used herein, refers to CN.

The terms “carbocycle”, and “carbocyclic”, as used herein, refers to asaturated or unsaturated ring in which each atom of the ring is carbon.The term carbocycle includes both aromatic carbocycles and non-aromaticcarbocycles. Non-aromatic carbocycles include both cycloalkane rings, inwhich all carbon atoms are saturated, and cycloalkene rings, whichcontain at least one double bond. “Carbocycle” includes 5-7 memberedmonocyclic and 8-12 membered bicyclic rings. Each ring of a bicycliccarbocycle may be selected from saturated, unsaturated and aromaticrings. Carbocycle includes bicyclic molecules in which one, two or threeor more atoms are shared between the two rings. The term “fusedcarbocycle” refers to a bicyclic carbocycle in which each of the ringsshares two adjacent atoms with the other ring. Each ring of a fusedcarbocycle may be selected from saturated, unsaturated and aromaticrings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, maybe fused to a saturated or unsaturated ring, e.g., cyclohexane,cyclopentane, or cyclohexene. Any combination of saturated, unsaturatedand aromatic bicyclic rings, as valence permits, is included in thedefinition of carbocyclic. Exemplary “carbocycles” include cyclopentane,cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene andadamantane. Exemplary fused carbocycles include decalin, naphthalene,1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane,4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene. “Carbocycles”may be substituted at any one or more positions capable of bearing ahydrogen atom.

The term “cycloalkyl”, as used herein, refers to the radical of asaturated aliphatic ring. In preferred embodiments, cycloalkyls havefrom 3-10 carbon atoms in their ring structure, and more preferably from5-7 carbon atoms in the ring structure. Suitable cycloalkyls includecycloheptyl, cyclohexyl, cyclopentyl, cyclobutyl and cyclopropyl.Cycloalkyl includes bicyclic molecules in which one, two or three ormore atoms are shared between the two rings. The term “fused cycloalkyl”refers to a bicyclic cycloalkyl in which each of the rings shares twoadjacent atoms with the other ring. The second ring of a fused bicycliccycloalkyl may be selected from saturated, unsaturated, and aromaticrings.

A “cycloalkenyl” group, as used herein, refers to a cyclic hydrocarboncontaining one or more double bonds. A “cycloalkynyl” group is a cyclichydrocarbon containing one or more triple bonds.

The terms “polycyclyl”, “polycycle”, and “polycyclic”, as used herein,refer to two or more rings (e.g., cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two ormore atoms are common to two adjoining rings, e.g., the rings are “fusedrings”. Each of the rings of the polycycle can be substituted orunsubstituted. In certain embodiments, each ring of the polycyclecontains from 3 to 10 atoms in the ring, preferably from 5 to 7.

The terms “halo” and “halogen”, as used herein, means halogen andincludes chloro, fluoro, bromo, and iodo.

The term “haloalkyl”, as used herein, means an alkyl group substitutedwith one or more halogens. When more than one halogen is present, thehalogens may be the same or different. For examples, haloalkyl groupsinclude, but are not limited to, fluoromethyl, difluoromethyl,trifluoromethyl, chlorodifluoromethyl, 2,2,2-trifluoroethyl,pentafluoroethyl, and the like.

The term “haloalkoxy”, as used herein, means an alkoxy group substitutedwith one or more halogens. When more than one halogen is present, thehalogens may be the same or different. For examples, haloalkyl groupsinclude, but are not limited to, difluoromethoxy, trifluoromethoxy,2,2,2-trifluoroethoxy, pentafluoroethoxy, and the like.

The term “heteroarylakyl”, as used herein, refers to an alkyl groupsubstituted with a heteroaryl group.

The term “heteroaryl” includes substituted or unsubstituted aromaticsingle ring structures, preferably 5- to 7-membered rings, morepreferably 5- to 6-membered rings, whose ring structures include atleast one heteroatom (e.g., O, N, or S), preferably one to four, or oneto 3 heteroatoms, more preferably one or two heteroatoms. When two ormore heteroatoms are present in a heteroaryl ring, they may be the sameor different. For examples, heteroaryl groups include, but are notlimited to, pyrrole, furan, thiophene, imidazole, tetrazole, oxazole,thiazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike. The term “heteroaryl” also include substituted or unsubstituted“polycyclic” ring systems having two or more cyclic rings in which twoor more carbons are common to two adjoining rings wherein at least oneof the rings is heteroaromatic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls.

The term “heteroatom”, as used herein, means an atom of any elementother than carbon or hydrogen. Exemplary heteroatoms include but are notlimited to nitrogen, oxygen, and sulfur.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. In certainembodiments, the ring structure is saturated, such as heterocycloalkyls;in other embodiments, the ring structure is unsaturated, such asheterocycloalkenyls or heterocycloalkynyls. The terms “heterocyclyl” and“heterocyclic” also include substituted or unsubstituted polycyclic ringsystems having two or more cyclic rings in which two or more carbons arecommon to two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like. In certainembodiments, the ring structure can have two cyclic rings. In someembodiments, the two cyclic rings can have two or more atoms in common,e.g., the rings are “fused rings.” Heterocyclyl groups include, but nolimited to, for example, piperidine, piperazine, pyrrolidine,morpholine, lactones, lactams, and the like.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of the disclosure, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, an alkylthio,an acyloxy, a phosphoryl, a phosphate, a phosphonate, an amino, anamido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl,an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, asulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromaticmoiety. It will be understood by those skilled in the art thatsubstituents can themselves be substituted, if appropriate. Accordingly,substituents can further include an acetamide, for example.

Unless specifically stated as “unsubstituted,” references to chemicalmoieties herein are understood to include substituted variants. Forexample, reference to an “alkyl” group or moiety implicitly includesboth substituted and unsubstituted variants. The term “unsubstituted”refers to that the specified group bears no substituents.

The term “optionally substituted”, as used herein, means thatsubstitution is optional and therefore it is possible for the designatedatom or moiety to be unsubstituted.

The term “ring” or “ring system”, unless context indicates otherwise,may include monocyclic rings or polycyclic rings, such as bicyclicrings. When the term ring refers to a polycyclic or bicyclic ring, eachring is independently selected from saturated or unsaturated, and eitheror both rings may contain one or more heteroatoms, preferably a total of0, 1, 2, 3 or 4 heteroatoms across the ring system.

A “pharmaceutically active metabolite” or “metabolite” refers to apharmacologically active product of metabolism/biochemical modificationof a compound described herein, e.g., a compound of Formula (I)(including Ia and Ib), (II) (including IIa and IIb), (III) (includingIIIa and IIIb), Formula II′ (including IIa′ and IIb′), and Formula III′(including IIIa′ and IIIb′) or salt thereof, under physiologicalconditions, e.g., through certain enzymatic pathway. For example, anoxidative metabolite is formed by oxidation of the parent compoundduring metabolism, such as the oxidation of a pyridine ring topyridine-N-oxide. In another example, an oxidative metabolite is formedby demethylation of a methoxy group to result in a hydroxyl group.

Compounds of this disclosure can also exist as various “solvates” or“hydrates.” A “hydrate” is a compound that exists in a composition withwater molecules. The composition can include water in stoichiometicquantities, such as a monohydrate or a dihydrate, or can include waterin random amounts. A “solvate” is a similar composition except that asolvent other that water, such as with methanol, ethanol,dimethylformamide, diethyl ether and the like replaces the water. Forexample, methanol or ethanol can form an “alcoholate,″” which can againbe stoichiometic or non-stoichiometric. Mixtures of such solvates orhydrates can also be prepared. The source of such solvate or hydrate canbe from the solvent of crystallization, inherent in the solvent ofpreparation or crystallization, or adventitious to such solvent.

The compounds of the disclosure, including their pharmaceuticallyacceptable salts and prodrugs, can exist as various polymorphs,pseudo-polymorphs, or in amorphous state. The term “polymorph”, as usedherein, refers to different crystalline forms of the same compound andother solid state molecular forms including pseudo-polymorphs, such ashydrates, solvates, or salts of the same compound. Different crystallinepolymorphs have different crystal structures due to a different packingof molecules in the lattice, as a result of changes in temperature,pressure, or variations in the crystallization process. Polymorphsdiffer from each other in their physical properties, such as x-raydiffraction characteristics, stability, melting points, solubility, orrates of dissolution in certain solvents. Thus crystalline polymorphicforms are important aspects in the development of suitable dosage formsin pharmaceutical industry.

In certain embodiments, a “pharmaceutically acceptable” substance issuitable for use in contact with cells, tissues or organs of animals orhumans without excessive toxicity, irritation, allergic response,immunogenicity or other adverse reactions, in the amount used in thedosage form according to the dosing schedule, and commensurate with areasonable benefit/risk ratio. In certain embodiments, a“pharmaceutically acceptable” substance that is a component of apharmaceutical composition is, in addition, compatible with the otheringredient(s) of the composition.

The terms “pharmaceutically acceptable excipient”, “pharmaceuticallyacceptable carrier” and “pharmaceutically acceptable diluent” encompass,without limitation, pharmaceutically acceptable inactive ingredients,materials, compositions and vehicles, such as liquid fillers, solidfillers, diluents, excipients, carriers, solvents and encapsulatingmaterials. Carriers, diluents and excipients also include allpharmaceutically acceptable dispersion media, coatings, buffers,isotonic agents, stabilizers, absorption delaying agents, antimicrobialagents, antibacterial agents, antifungal agents, adjuvants, and so on.Except insofar as any conventional excipient, carrier or diluent isincompatible with the active ingredient, the present disclosureencompasses the use of conventional excipients, carriers and diluents inpharmaceutical compositions. See, e.g., Remington: The Science andPractice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins(Philadelphia, Pa., 2005); Handbook of Pharmaceutical Excipients, 5thEd., Rowe et al., Eds., The Pharmaceutical Press and the AmericanPharmaceutical Association (2005); Handbook of Pharmaceutical Additives,3rd Ed., Ash and Ash, Eds., Gower Publishing Co. (2007); andPharmaceutical Preformulation and Formulation, Gibson, Ed., CRC PressLLC (Boca Raton, Fla., 2004). A “pharmaceutically acceptable salt” is asalt of a compound that is suitable for pharmaceutical use, includingbut not limited to metal salts (e.g., sodium, potassium, magnesium,calcium, etc.), acid addition salts (e.g., mineral acids, carboxylicacids, etc.), and base addition salts (e.g., ammonia, organic amines,etc.).

“Pharmaceutically acceptable salt” or “salt” is used herein to refer toan agent or a compound according to the disclosure that is atherapeutically active, non-toxic base and acid salt form of thecompounds. The acid addition salt form of a compound that occurs in itsfree form as a base can be obtained by treating said free base form withan appropriate acid such as an inorganic acid, for example, a hydrohalicsuch as hydrochloric or hydrobromic, sulfuric, nitric, phosphoric andthe like; or an organic acid, such as, for example, acetic,hydroxyacetic, propanoic, lactic, pyruvic, malonic, succinic, maleic,fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic,benzenesulfonic, p-toluenesulfonic, cyclic, salicylic, p-aminosalicylic,pamoic and the like. See, e.g., WO 01/062726. Some pharmaceuticallyacceptable salts listed by Berge et al., Journal of PharmaceuticalSciences, 66: 1-19 (1977), incorporated herein by reference in itsentirety.

Compounds containing acidic protons may be converted into theirtherapeutically active, non-toxic base addition salt form, e. g. metalor amine salts, by treatment with appropriate organic and inorganicbases. Appropriate base salt forms include, for example, ammonium salts,alkali and earth alkaline metal salts, e. g., lithium, sodium,potassium, magnesium, calcium salts and the like, salts with organicbases, e. g. N-methyl-D-glucamine, hydrabamine salts, and salts withamino acids such as, for example, arginine, lysine and the like.Conversely, said salt forms can be converted into the free forms bytreatment with an appropriate base or acid. Compounds and their saltscan be in the form of a solvate, which is included within the scope ofthe present disclosure. Such solvates include for example hydrates,alcoholates and the like. See, e.g., WO 01/062726.

Other examples of pharmaceutically acceptable salts include, but are notlimited to, camsylate, sulfates, pyrosulfates, bisulfates, sulfites,bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates,metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates,propionates, decanoates, caprylates, acrylates, formates, isobutyrates,caproates, heptanoates, propiolates, oxalates, malonates, succinates,suberates, sebacates, fumarates, maleates, butyne-1,4-dioates,hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates,dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates,sulfonates, methylsulfonates, propylsulfonates, besylates,xylenesulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates,phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates,γ-hydroxybutyrates, glycolates, tartrates, and mandelates. Lists ofother suitable pharmaceutically acceptable salts are found inRemington's Pharmaceutical Sciences, 17th Edition, Mack PublishingCompany, Easton, Pa., 1985.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentdisclosure.

The term “pharmaceutical composition” refers to a composition suitablefor pharmaceutical use in a subject animal, including humans andmammals, e.g., combined with one or more pharmaceutically acceptablecarriers, excipients or solvents. Such a composition may also containdiluents, fillers, salts, buffers, stabilizers, solubilizers, and othermaterials well known in the art. In certain embodiments, apharmaceutical composition encompasses a composition comprising theactive ingredient(s), and the inert ingredient(s) that make up theexcipient, carrier or diluent, as well as any product that results,directly or indirectly, from combination, complexation or aggregation ofany two or more of the ingredients, or from dissociation of one or moreof the ingredients, or from other types of reactions or interactions ofone or more of the ingredients. Accordingly, the pharmaceuticalcompositions of the present disclosure encompass any composition made byadmixing a compound of the disclosure and one or more pharmaceuticallyacceptable excipient(s), carrier(s) and/or diluent(s).

Accordingly, the pharmaceutical compositions of the present disclosureencompass any composition made by admixing a compound of the disclosureand one or more pharmaceutically acceptable excipient(s), carrier(s)and/or diluent(s).

The disclosure further provides pharmaceutical compositions comprisingone or more compounds of the disclosure together with a pharmaceuticallyacceptable carrier or excipient. Compounds or pharmaceuticalcompositions of the disclosure may be used in vitro or in vivo.

Isomerism and Tautomerism in Described Compounds

Tautomerism

Within the present disclosure it is to be understood that a compounddescribed herein or a salt thereof may exhibit the phenomenon oftautomerism whereby two chemical compounds that are capable of facileinterconversion by exchanging a hydrogen atom between two atoms, toeither of which it forms a covalent bond. Since the tautomeric compoundsexist in mobile equilibrium with each other they may be regarded asdifferent isomeric forms of the same compound. It is to be understoodthat the formulae drawings within this specification can represent onlyone of the possible tautomeric forms. However, it is also to beunderstood that the disclosure encompasses any tautomeric form, and isnot to be limited merely to any one tautomeric form utilized within theformulae drawings. The formulae drawings within this specification canrepresent only one of the possible tautomeric forms and it is to beunderstood that the specification encompasses all possible tautomericforms of the compounds drawn not just those forms which it has beenconvenient to show graphically herein. For example, tautomerism may beexhibited by a pyrazolyl group bonded as indicated by the wavy line.While both substituents would be termed a 4-pyrazolyl group, it isevident that a different nitrogen atom bears the hydrogen atom in eachstructure.

Such tautomerism can also occur with substituted pyrazoles such as3-methyl, 5-methyl, or 3,5-dimethylpyrazoles, and the like. Anotherexample of tautomerism is amido-imido (lactam-lactim when cyclic)tautomerism, such as is seen in heterocyclic compounds bearing a ringoxygen atom adjacent to a ring nitrogen atom. For example, theequilibrium:

is an example of tautomerism. Accordingly, a structure depicted hereinas one tautomer is intended to also include the other tautomer.Optical Isomerism

It will be understood that when compounds of the present disclosurecontain one or more chiral centers, the compounds may exist in, and maybe isolated as pure enantiomeric or diastereomeric forms or as racemicmixtures. The present disclosure therefore includes any possibleenantiomers, diastereomers, racemates in their pure forms or mixturesthereof, and salts thereof, of the compounds of the disclosure.

The isomers resulting from the presence of a chiral center comprise apair of non-superimposable isomers that are called “enantiomers.” Singleenantiomers of a pure compound are optically active, i.e., they arecapable of rotating the plane of plane polarized light. Singleenantiomers are designated according to the Cahn-Ingold-Prelog system.The priority of substituents is ranked based on atomic weights, a higheratomic weight, as determined by the systematic procedure, having ahigher priority ranking. Once the priority ranking of the four groups isdetermined, the molecule is oriented so that the lowest ranking group ispointed away from the viewer. Then, if the descending rank order of theother groups proceeds clockwise, the molecule is designated (R) and ifthe descending rank of the other groups proceeds counterclockwise, themolecule is designated (S). In the example in Scheme 14, theCahn-Ingold-Prelog ranking is A>B>C>D. The lowest ranking atom, D isoriented away from the viewer.

In certain embodiments, the therapeutic preparation may be enriched toprovide predominantly one enantiomer of a compound (e.g., of formula(I), (Ia), or (Ib)). An enantiomerically enriched mixture may comprise,for example, at least 60 mol percent of one enantiomer, or morepreferably at least 75, 90, 95, or even 99 mol percent. In certainembodiments, a compound of the invention may have greater than 30% ee,40% ee, 50% ee, 60% ee, 70% ee, 80% ee, 90% ee, or even 95% or greateree. In certain embodiments, the compound enriched in one enantiomer issubstantially free of the other enantiomer, wherein substantially freemeans that the substance in question makes up less than 10%, or lessthan 5%, or less than 4%, or less than 3%, or less than 2%, or less than1% as compared to the amount of the other enantiomer, e.g., in thecomposition or compound mixture. For example, if a composition orcompound mixture contains 98 grams of a first enantiomer and 2 grams ofa second enantiomer, it would be said to contain 98 mol percent of thefirst enantiomer and only 2% of the second enantiomer.

In certain embodiments, compounds of the disclosure may have more thanone stereocenter. In certain such embodiments, compounds of thedisclosure may be enriched in one or more diastereomer. For example, acompound of the disclosure may have greater than 30% de, 40% de, 50% de,60% de, 70% de, 80% de, 90% de, or even 95% or greater de.

Isolated optical isomers may be purified from racemic mixtures bywell-known chiral separation techniques, such as but not limited to,normal and reverse phase chromatography, and crystallization. Accordingto one such method, a racemic mixture of a compound of the disclosure,or a chiral intermediate thereof, is separated using a chiral salt orcarried out on a Chiralcell OD column. The column is operated accordingto the manufacturer's instructions.

Isolated optical isomers (enantiomerically pure compounds) can also beprepared by the use of chiral intermediates or catalysts in synthesis.When a chiral synthetic intermediate is used, the optical center (chiralcenter) can be preserved without racemization throughout the remainderof the preparative procedure, as is well known in the art. Chiralcatalyst can be used to impart at least some degree of enantiomericpurity to products of reactions catalyzed by the chiral catalyst. And,in some cases, compounds having at least some degree of enantiomericenrichment can be obtained by physical processes such as selectivecrystallization of salts or complexes formed with chiral adjuvants.

A variety of compounds in the present disclosure may exist in particulargeometric or stereoisomeric forms. The present disclosure takes intoaccount all such compounds, including tautomers, cis- and trans-isomers,R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, theracemic mixtures thereof, and other mixtures thereof, as being coveredwithin the scope of this disclosure. All tautomeric forms areencompassed in the present disclosure. Additional asymmetric carbonatoms may be present in a substituent such as an alkyl group. All suchisomers, as well as mixtures thereof, are intended to be included inthis disclosure, unless the stereochemistry or isomeric form isspecifically indicated.

Rotational Isomerism

It is understood that due to chemical properties (i.e., resonancelending some double bond character to the C—N bond) of restrictedrotation about the amide bond linkage (as illustrated below) it ispossible to observe separate rotamer species and even, under somecircumstances, to isolate such species (see below). It is furtherunderstood that certain structural elements, including steric bulk orsubstituents on the amide nitrogen, may enhance the stability of arotamer to the extent that a compound may be isolated as, and existindefinitely, as a single stable rotamer. The present disclosuretherefore includes any possible stable rotamers of formula (I) which arebiologically active in the treatment of cancer or other proliferativedisease states.

Regioisomerism

The compounds of the disclosure have a particular spatial arrangement ofsubstituents on the aromatic rings, which are related to the structureactivity relationship demonstrated by the compound class. Often suchsubstitution arrangement is denoted by a numbering system; however,numbering systems are often not consistent between different ringsystems. In six-membered aromatic systems, the spatial arrangements arespecified by the common nomenclature “para” for 1,4-substitution, “meta”for 1,3-substitution and “ortho” for 1,2-substitution as shown below.

In various embodiments, the compound or set of compounds, such as areused in the disclosed methods, can be any one of any of the combinationsand/or sub-combinations of the above-listed embodiments.

Cis-, Trans-Isomerism or E/Z Isomerism

In some embodiments, the compounds of the disclosure may contain adouble bond. It is understood that cis/trans isomers are configurationalisomers having different orientation at the double bond. In certain suchembodiments, the compounds of this disclosure can be in either cis- ortrans-formation. In the present disclosure, the term “cis” isequivalently used for “Z” and vice versa “trans” for ‘E” and vice versa.

A “patient,” “subject,” or “individual” are used interchangeably andrefer to both mammals and non-mammals. Mammals include, for example,humans; non-human primates either a human or a non-human animal. Theseterms include mammals, such as humans, non-human primates, e.g. apes andmonkeys; and non-primates, e.g. mice, rats, rabbits, dogs, cats, cattle,horses, sheep, and goats. In certain embodiments, the patient or subjectis a human patient or subject, such as a human patient having acondition associated with SHMT activity and in need of treatment.

“SHMT” refers to serine hydroxymethyltransferase. Such enzymes are knownand, in mammals, both SHMT1 and SHMT2 are expressed and active.Exemplary SHMTs include mammalian SHMT1 and SHMT2, such as human SHMT1and SHMT2. Further structural information regarding human SHMT1 can befound at NCBI entrez ID number 6470. Further structural informationregarding human SHMT2 can be found at NCBI entrez ID number 6472.

“Inhibitor” as used herein refers to any molecule that is capable ofinteracting directly or indirectly with another molecule (e.g., anenzyme or receptor) and causing a decrease in a biological activity ofthat other molecule. In certain embodiments, the compounds of thepresent disclosure inhibit mammalian SHMT enzyme activity (e.g., SHMT1and/or SHMT2). In certain embodiments the inhibitors are selectiveinhibitors of SHMT enzyme activity (e.g., SHMT1 and/or SHMT2). Incertain embodiments, the inhibitors bind to SHMT1 and/or SHMT2 (e.g.,bind to the enzyme). In certain embodiments, the SHMT inhibitor is aninhibitor of SHMT2 and does not inhibit or inhibits with a significantlylower IC₅₀ an activity of SHMT1. In certain embodiments, the SHMTinhibitor inhibits an activity of SHMT2 and, optionally, SHMT1. Incertain embodiments, the SHMT inhibitor inhibits both SHMT1 and SHMT2(e.g., either with approximately the same IC₅₀ or within 2, 3 or4-fold). In certain embodiments, the compounds of the disclosure (suchas compounds of Formulae (I)-(IX) (including compounds of Formulae(I)-(IX), and pharmaceutically acceptable salts thereof, as well as theindividual compounds disclosed herein) are used as inhibitors of SHMTactivity (e.g., enzyme activity). In certain embodiments, compounds ofthe disclosure are SHMT inhibitors, such as selective SHMT inhibitors(SHMT1 and/or SHMT2). It should be noted that a compound may becharacterized as an SHMT1 and/or SHMT2 inhibitor by evaluation in an invitro assay. This gives an accurate characterization. However, in vivo,the compound's mechanism of action may be primarily via its effect onone but not both enzymes. For example, when used in a subject or cellline deficient in SHMT2, the compound's effect on cell proliferation maybe primarily through its effect as an SHMT1 inhibitor. Similarly, insome systems, a compound may have poor penetration or accessibility tothe mitochondria, and thus, the effect of the compound may be primarilythrough its effect on SHMT1 despite its high intrinsic activity againstSHMT2. Regardless of the particular mechanism of action at play in anyparticular in vivo system, inhibitors may be characterized as SHMT2and/or SHMT1 inhibitors based on activity in one or more in vitroassays, as described herein.

In certain embodiments, by “SHMT activity” is meant a native function ofa mammalian SHMT enzyme, such its native enzymatic activity. In certainembodiments, SHMT activity refers to the function of mammalian SHMT tocatalyze a reversible reaction converting serine to glycine. In certainembodiments, SHMT activity refers to the function of mammalian SHMT tocatalyze a reversible reaction converting serine to glycine withconcurrent methylene-tetrahydrofolate (meTHF) generation. In certainembodiments, SHMT activity refers to the generation of 1C units. SHMTactivity may be assayed or evaluated in numerous ways, such as isdescribed herein. SHMT activity may be evaluated by evaluating serineflux and/or folate metabolism, such as mitochondrial serine flux,glycine synthesis, NADPH generation, generation and excretion of formateor mitochondrial folate metabolism.

The term “compounds of the disclosure” refers to any of the compoundsdescribed herein based on any combination of structural and/orfunctional features, including compounds of Formulae (I)-(IX), whereinthe variables are defined as provided herein, as well as to any of thespecific compounds described herein. The term “compounds of thedisclosure” refers, unless context indicates otherwise, to salts of suchcompounds, such as pharmaceutically acceptable salts. In certainembodiments, compounds of the disclosure are capable of inhibiting SHMTactivity, such as enzyme activity. In certain embodiments, compounds ofthe disclosure are inhibitors of SHMT2 and/or SHMT1. In certainembodiments, compounds of the disclosure are selective inhibitors ofSHMT (e.g., SHMT1 and/or 2). In certain embodiments, compounds of thedisclosure are dual inhibitors of SHMT1 and SHMT2. In certainembodiments, compounds of the disclosure are selective for SHMT overMTHFD2 and/or DHFR and/or TS. For example, in certain embodiments,compounds of the disclosure either do not inhibit or inhibit one or moreof MTHFD2, DHFR, FH, TS and/or another protein involved in mitochondrialfolate metabolism with an IC50 at least 25 fold, at least 50 fold, atleast 75 fold, at least 100 fold, at least 200 fold, at least 500 fold,at least 1000 fold, or greater than 1000 fold less than that for SHMT2and/or SHMT1.

In certain embodiments, compounds of the disclosure include compoundsprovided as a pharmaceutical composition.

Compounds of the disclosure also include tautomeric forms, such asketo-enol tautomers, prototropic tautomers, and the like, for exampleannular tautomers wherein a proton can occupy two or more positions on aheteroaryl system. Tautomeric forms can be in equilibrium or stericallylocked into one form by appropriate substitution. It is understood thatthe various tautomeric forms are within the scope of the compounds ofthe present disclosure.

Compounds of the disclosure also include all isotopes of atoms occurringin the intermediates and/or final compounds. Isotopes include thoseatoms having the same atomic number but different mass numbers. Forexample, isotopes of hydrogen include deuterium and tritium.

Compounds of Formulae (I)-(IX) (and the other compounds of thedisclosure) have one or more chiral centers and therefore can exist asenantiomers and/or diastereomers. Compounds of Formulae (I)-(IX) (andthe other compounds of the disclosure) may also exist as stereoisomers,for example atropisomers, resulting from hindered rotation about asingle bond. The compound of the disclosure are understood to extend to,and embrace all such enantiomers, diastereomers, atropisomers,stereoisomers, and mixtures thereof, including but not limited toracemates. Formulae (I)-(IX) (and the other compounds of the disclosure)used throughout this disclosure are intended to represent all individualstereoisomers and mixtures thereof, unless stated or shown otherwise.

“Treating” a condition or patient refers to taking steps to obtainbeneficial or desired results, including clinical results. Beneficial ordesired clinical results include, but are not limited to, alleviation,amelioration, or slowing the progression, of one or more symptomsassociated with a condition, such as cancer. Exemplary beneficialclinical results are described herein.

“Administering” or “administration of” a substance, a compound or anagent to a subject can be carried out using one of a variety of methodsknown to those skilled in the art. For example, a compound or an agentcan be administered, intravenously, arterially, intradermally,intramuscularly, intraperitoneally, subcutaneously, ocularly,sublingually, orally (by ingestion), intranasally (by inhalation),intraspinally, intracerebrally, and transdermally (by absorption, e.g.,through a skin duct). A compound or agent can also appropriately beintroduced by rechargeable or biodegradable polymeric devices or otherdevices, e.g., patches and pumps, or formulations, which provide for theextended, slow or controlled release of the compound or agent.Administering can also be performed, for example, once, a plurality oftimes, and/or over one or more extended periods. In some aspects, theadministration includes both direct administration, includingself-administration, and indirect administration, including the act ofprescribing a drug. For example, as used herein, a physician whoinstructs a patient to self-administer a drug, or to have the drugadministered by another and/or who provides a patient with aprescription for a drug is administering the drug to the patient. When amethod is part of a therapeutic regimen involving more than one agent ortreatment modality, the disclosure contemplates that the agents may beadministered at the same or differing times and via the same ordiffering routes of administration.

Appropriate methods of administering a substance, a compound or an agentto a subject will also depend, for example, on the age of the subject,whether the subject is active or inactive at the time of administering,whether the subject is cognitively impaired at the time ofadministering, the extent of the impairment, and the chemical andbiological properties of the compound or agent (e.g. solubility,digestibility, bioavailability, stability and toxicity).

A “therapeutically effective amount” or a “therapeutically effectivedose” of a drug or agent is an amount of a drug or an agent that, whenadministered to a subject will have the intended therapeutic effect,sufficient to show a meaningful patient benefit, e.g., treatment,healing, inhibition or amelioration of a physiological response orcondition, etc. The full therapeutic effect does not necessarily occurby administration of one dose, and may occur only after administrationof a series of doses. Thus, a therapeutically effective amount may beadministered in one or more administrations. The precise effectiveamount needed for a subject will depend upon, for example, the subject'ssize, health and age, the nature and extent of disease, the therapeuticsor combination of therapeutics selected for administration, and the modeof administration. The skilled worker can readily determine theeffective amount for a given situation by routine experimentation.

C. Compounds

The present disclosure provides a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein:

-   R⁰, R¹ and R² are each independently selected from the group    consisting of —H, halogen (such as F, Br, or Cl), hydroxyl, nitro,    nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹²,    —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹,    —NS(O)₂R¹², substituted or unsubstituted alkyl (such as C₁-C₈ alkyl    or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl), substituted or    unsubstituted alkenyl (such as C₂-C₈ alkenyl), substituted or    unsubstituted alkynyl (such as C₂-C₈ alkynyl), substituted or    unsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl; e.g.,    cyclopropyl or cyclobutyl), substituted or unsubstituted    heterocyclyl, substituted or unsubstituted aryl, substituted or    unsubstituted heteroaryl, substituted or unsubstituted arylalkyl,    substituted or unsubstituted heteroarylalkyl, substituted or    unsubstituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl;    e.g., trifluoromethyl), and substituted or unsubstituted haloalkoxy    (such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy); provided that, at    least one of R⁰, R¹ and R² is selected from the group consisting of    substituted or unsubstituted alkenyl (such as C₂-C₈ alkenyl), and    substituted or unsubstituted alkynyl (such as C₂-C₈ alkynyl);-   R³ is selected from the group consisting of —H, halogen, hydroxyl,    nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹²,    —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹,    —NS(O)₂R¹², substituted or unsubstituted alkyl (such as C₁-C₈ alkyl    or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl), substituted or    unsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl; e.g.,    cyclopropyl or cyclobutyl), substituted or unsubstituted    heterocyclyl, substituted or unsubstituted aryl, substituted or    unsubstituted heteroaryl, substituted or unsubstituted arylalkyl,    substituted or unsubstituted heteroarylalkyl, substituted or    unsubstituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl;    e.g., trifluoromethyl), and substituted or unsubstituted haloalkoxy    (such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy);-   R⁴ is selected from the group consisting of —H, substituted or    unsubstituted alkyl (such as C₁-C₈ alkyl; e.g., methyl, ethyl, or    iso-propyl), substituted or unsubstituted cycloalkyl (such as C₃-C₇    cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl,    substituted or unsubstituted arylalkyl, and substituted or    unsubstituted heteroarylalkyl;-   R⁵, R⁶ and R⁷ are each independently selected from the group    consisting of —H, —C(O)R¹¹, substituted or unsubstituted alkyl (such    as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl;    e.g., cyclopropyl or cyclobutyl), substituted or unsubstituted aryl,    substituted or unsubstituted heteroaryl, substituted or    unsubstituted arylalkyl, and substituted or unsubstituted    heteroarylalkyl; or R⁵ is selected from any of the foregoing and R⁶    and R⁷ taken together with the nitrogen atom to which they are    attached form a substituted or unsubstituted 3-6 membered ring;-   each occurrence of R¹¹ is independently selected from the group    consisting of substituted or unsubstituted alkyl (such as C₁-C₈    alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl;    e.g., cyclopropyl or cyclobutyl), substituted or unsubstituted    heterocyclyl, substituted or unsubstituted aryl, and substituted or    unsubstituted heteroaryl; and-   each occurrence of R¹⁰ and R¹² is independently selected from the    group consisting of —H, substituted or unsubstituted alkyl (such as    C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl;    e.g., cyclopropyl or cyclobutyl), substituted or unsubstituted    heterocyclyl, substituted or unsubstituted aryl, and substituted or    unsubstituted heteroaryl.

In some embodiments, the compound of this disclosure is represented byFormula (Ia):

or a pharmaceutically acceptable salt there.

In certain embodiments, the compound of this disclosure is representedby Formula (Ib):

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, R⁰, R¹ andR² are each independently selected from the group consisting of —H,halogen, hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹²,—OR¹¹, —C(O)OR¹², —C(O)R¹⁰R¹², —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹,—NS(O)₂R¹², substituted or unsubstituted alkyl (such as C₁-C₈ alkyl orC₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl), substituted orunsubstituted alkenyl (such as C₂-C₈ alkenyl), substituted orunsubstituted alkynyl (such as C₂-C₈ alkynyl), substituted orunsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl; e.g., cyclopropyl orcyclobutyl), substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted haloalkyl (such as C₁-C₈haloalkyl or C₁-C₆ haloalkyl; e.g., trifluoromethyl), and substituted orunsubstituted haloalkoxy (such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy);provided that, at least one of R⁰, R¹ and R² is independently selectedfrom the group consisting of substituted or unsubstituted alkenyl, andsubstituted or unsubstituted alkynyl.

In certain embodiments of any of the foregoing or following, one of R¹and R² is selected from the group consisting of substituted orunsubstituted alkenyl (such as C₂-C₈ alkenyl), and substituted orunsubstituted alkynyl (such as C₂-C₈ alkynyl); the other isindependently selected from the group consisting of —H, halogen,hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹²,substituted or unsubstituted alkyl (such as C₁-C₈ alkyl or C₁-C₆ alkyl;e.g., methyl, ethyl, or iso-propyl), substituted or unsubstitutedalkenyl (such as C₂-C₈ alkenyl), substituted or unsubstituted alkynyl(such as C₂-C₈ alkynyl), substituted or unsubstituted cycloalkyl (suchas C₃-C₇ cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl;e.g., trifluoromethyl), and substituted or unsubstituted haloalkoxy(such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy).

In other embodiments of any of the foregoing or following, one of R¹ andR² is selected from the group consisting of substituted or unsubstitutedalkenyl (such as C₂-C₈ alkenyl), and substituted or unsubstitutedalkynyl (such as C₂-C₈ alkynyl); the other is independently selectedfrom the group consisting of —H, halogen, hydroxyl, nitro, nitrile,—OR¹¹, substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl;e.g., trifluoromethyl), and substituted or unsubstituted haloalkoxy(such as C₂-C₈ haloalkoxy or C₁-C₆ haloalkoxy).

In certain embodiments of any of the foregoing or following, one of R¹and R² is substituted or unsubstituted alkynyl (such as C₂-C₈ alkynyl),the other is not. In other embodiments, both R¹ and R² are substitutedor unsubstituted alkynyl (such as C₂-C₈ alkynyl).

In some embodiments of any of the foregoing of following, one of R¹ andR² is substituted or unsubstituted alkenyl (such as C₂-C₈ alkenyl), theother is not. In other embodiments, both R¹ and R² are substituted orunsubstituted alkenyl (such as C₂-C₈ alkenyl).

In certain embodiments of any of the foregoing or following, R¹ issubstituted or unsubstituted alkynyl and R² is not a substituted orunsubstituted alkynyl (such as C₂-C₈ alkynyl). In other embodiments, R²is substituted or unsubstituted alkynyl and R¹ is not a substituted orunsubstituted alkynyl (such as C₂-C₈ alkynyl).

In certain embodiments of any of the foregoing or following, R¹ issubstituted or unsubstituted alkenyl and R² is not a substituted orunsubstituted alkenyl (such as C₂-C₈ alkenyl). In other embodiments, R²is substituted or unsubstituted alkenyl and R¹ is not a substituted orunsubstituted alkenyl (such as C₂-C₈ alkenyl).

In certain embodiments of any of the foregoing or following, the alkenyl(such as C₂-C₈ alkenyl) or alkynyl (such as C₂-C₈ alkynyl), whensubstituted, is substituted with one or more substituents independentlyselected from the group consisting of halogen, hydroxyl, nitro, nitrile,—SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², optionallysubstituted aryl, and optionally substituted heteroaryl comprising 1-4 Natoms; or two of the substituents together with the atoms to which theyare attached form an optionally substituted ring.

In certain such embodiments of any of the foregoing or following,alkenyl (such as C2-C8 alkenyl) or alkynyl (such as C2-C8 alkynyl), whensubstituted, is substituted with one or more substituents independentlyselected from the group consisting of OH, halogen, —OR11, —C(O)OR 12,—C(O)NR10R12, —NR10R12, optionally substituted aryl, and optionallysubstituted heteroaryl comprising 1-4 N atoms; or two of thesubstituents together with the atoms to which they are attached form anoptionally substituted ring.

In certain embodiments of any of the foregoing or following, R0 isselected from the group consisting of hydroxyl, —S(O)2R11,—S(O)2NR10R12, —OR11, —C(O)NR10R12, —NR 10R12, —N(R12)C(O)R11, and—NS(O)2R12. In certain such embodiments, R0 is H.

In certain embodiments of any of the foregoing or following, R³ isselected from the group consisting of methyl, ethyl, propyl, isopropyl,cyclopropyl, and cyclobutyl. In certain such embodiments, R3 is selectedfrom the group consisting of isopropyl, cyclopropyl, and cyclobutyl. Insome embodiments, R3 is isopropyl.

In certain embodiments of any of the foregoing or following, R4 isselected from the group consisting of methyl, ethyl, isopropyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, and benzyl. Incertain such embodiments, R4 is methyl or isopropyl. In someembodiments, R4 is methyl.

In certain embodiments of any of the foregoing or following, R5, R6, andR7 are each independently selected from the group consisting of —H,methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, phenyl, benzyl, and —COCH3. In certain such embodiments, R5,R6 and R7 are each independently selected from the group consisting of—H, methyl, phenyl, and —COCH3. In some embodiments, R5 and R6 are eachindependently selected from the group consisting of —H, methyl, andphenyl.

In certain embodiments of any of the foregoing or following, R7 is —H.

In certain embodiments of any of the foregoing or following, R5 isselected from the group consisting of —H, methyl, ethyl, isopropyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, benzyl, and—COCH3; and R6 and R7 taken together with the nitrogen atom to whichthey are attached form a substituted or unsubstituted ring selected fromthe group consisting of:

In certain embodiments of any of the foregoing or following, R⁵ isselected from the group consisting of —H, methyl, phenyl, and —COCH₃;and R⁶ and R⁷ taken together with the nitrogen atom to which they areattached form a substituted or unsubstituted ring selected from thegroup consisting of:

In certain embodiments of any of the foregoing or following, R⁰ isselected from the group consisting of —H, halogen, hydroxyl, nitro,nitrile, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl (such asC₁-C₈ alkyl; e.g., methyl, ethyl, or iso-propyl), substituted orunsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl; e.g., cyclopropyl orcyclobutyl), substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted haloalkyl (such as C₁-C₈haloalkyl or C₁-C₆ haloalkyl; e.g., trifluoromethyl), and substituted orunsubstituted haloalkoxy (such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy);

one of R¹ and R² is substituted or unsubstituted alkenyl (such as C₂-C₈alkenyl), or substituted or unsubstituted alkynyl (such as C₂-C₈alkynyl); the other is independently selected from the group consistingof —H, halogen, hydroxyl, nitro, nitrile, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹²,—OR¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substitutedor unsubstituted alkyl (such as C₁-C₈ alkyl; e.g., methyl, ethyl, oriso-propyl), unsubstituted or unsubstituted alkenyl (such as C₂-C₈alkenyl), substituted or unsubstituted alkynyl (such as C₂-C₈ alkynyl),substituted or unsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl; e.g.,cyclopropyl or cyclobutyl), substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstituted haloalkyl(such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl); e.g., trifluoromethyl),and substituted or unsubstituted haloalkoxy (such as C₁-C₈ haloalkoxy orC₁-C₆ haloalkoxy);

R³ is selected from the group consisting of methyl, ethyl, propyl,isopropyl, cyclopropyl, and cyclobutyl;

R⁴ is selected from the group consisting of methyl, ethyl, isopropyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl and benzyl; and

R⁵, R⁶ and R⁷ are each independently selected from the group consistingof —H, methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, phenyl, benzyl, and —COCH₃.

In certain embodiments of any of the foregoing of following, R⁰ is —H;

one of R¹ and R² is substituted or unsubstituted alkenyl (such as C₂-C₈alkenyl), or substituted or unsubstituted alkynyl (such as C₂-C₈alkenyl); the other is independently selected from the group consistingof —H, halogen, hydroxyl, nitro, nitrile, —OR¹¹, substituted orunsubstituted alkyl (such as C₁-C₈ alkyl C₁-C₆ alkyl); e.g., methyl,ethyl, or iso-propyl), substituted or unsubstituted alkenyl (such asC₂-C₈ alkenyl), substituted or unsubstituted alkynyl (such as C₂-C₈alkynyl), substituted or unsubstituted haloalkyl (such as C₁-C₈haloalkyl or C₁-C₆ haloalkyl); e.g., trifluoromethyl), or substituted orunsubstituted haloalkoxy (such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy);

R³ is selected from the group consisting of isopropyl, cyclopropyl, andcyclobutyl;

R⁴ is methyl or isopropyl; and

R⁵, R⁶ and R⁷ are each independently selected from the group consistingof —H, alkyl (such as C₁-C₈ alkyl C₁-C₆ alkyl); e.g., methyl, ethyl, oriso-propyl), phenyl, and —COCH₃.

In certain embodiments, R⁰ is —H;

one of R¹ and R² is substituted or unsubstituted alkenyl (such as C₂-C₈alkenyl), or substituted or unsubstituted alkynyl (such as C₂-C₈alkynyl); the other is independently selected from the group consistingof —H, methoxy, fluoro, chloro, bromo, hydroxyl, nitro, nitrile, alkyl,—CCl₃, and —CF₃;

R³ is cyclobutyl or iso-propyl;

R⁴ is methyl;

R⁵ and R⁶ are each independently selected from the group consisting of—H, alkyl (such as C₁-C₆alkyl; e.g., methyl, ethyl, or iso-propyl), andphenyl; and

R⁷ is H.

In certain embodiments of any of the foregoing or following, R⁰ isselected from the group consisting of —H, hydroxyl, —S(O)₂R¹¹,—S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, and—NS(O)₂R¹²;

one of R¹ and R² is substituted or unsubstituted alkynyl (such as C₂-C₈alkynyl); the other is nitro, —Cl, —OCH₃, or —CF₃; R³ is iso-propyl; R⁴is methyl; and R⁵, R⁶, and R⁷ are H.

The disclosure contemplates compounds have any combination of any of theforegoing or following structural and/or functional characteristics.

In certain embodiments, this disclosure provides a compound of Formula(II):

or a pharmaceutically acceptable salt thereof, wherein

R², R³, R⁴, R⁵, R⁶, and R⁷ are as defined herein;

W represents —CR¹⁶═CR¹⁶— or —C≡C—;

n is 0, 1, 2, 3, or 4;

R¹³, R¹⁴, and R¹⁵ are independently selected from the group consistingof hydrogen, —OH, halogen, optionally substituted alkyl (such as C₁-C₈alkyl or C₁-C₆ alkyl); e.g., methyl, ethyl, or iso-propyl), optionallysubstituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl; e.g.,trifluoromethyl), —OR^(a), —OC(O)R^(b), —C(O)NR^(a)R^(b), and—NR^(a)R^(b); or R¹³ and R¹⁴ together with the atom to which they areattached form a 4-7 membered heterocyclic ring comprising 1 or 2heteroatoms selected from the group consisting of NR, O, S, or SO, orSO₂; wherein the heterocyclic ring is optionally substituted with one ormore substituents independently selected from the group consisting ofoxo and optionally substituted alkyl; and R¹⁶, R^(a) and R^(b),independently at each occurrence, are H or optionally substituted alkyl(such as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, oriso-propyl).

In certain embodiments, the compound of Formula (II) can be representedby Formula (IIa) or (IIb):

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, W is—CR¹⁶CR¹⁶ in cis- or trans-formation. In other embodiments, W is —C≡C—.

In certain embodiments of any of the foregoing of following, R¹³, R¹⁴,and R¹⁵ are independently selected from the group consisting ofhydrogen, —OH, halogen, optionally substituted alkyl (such as C₁-C₈alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl), optionallysubstituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl; e.g.,trifluoromethyl), —OR^(a), —OC(O)R^(b), —C(O)NR^(a)R^(b), and—NR^(a)R^(b), and wherein Re and R^(b), independently at eachoccurrence, are H or optionally substituted alkyl ((such as C₁-C₈ alkylor C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl).

In certain embodiments of any of the foregoing or following, n is 0; R¹³and R¹⁴ together with the atom to which they are attached form a 4-7membered heterocyclic ring comprising 1 or 2 heteroatoms selected fromthe group consisting of NR^(a), and O; wherein the heterocyclic ring isoptionally substituted with one or more substituents independentlyselected from the group consisting of oxo and optionally substitutedalkyl; and R¹⁵ is H.

The disclosure contemplates compounds have any combination of any of theforegoing or following structural and/or functional characteristics.

In certain embodiments of any of the foregoing or following, thisdisclosure provides a compound of Formula (III):

or a pharmaceutically acceptable salt thereof,

wherein

R², R³, R⁴, R⁵, R⁶, and R⁷ are as defined herein;

W represents —CR¹⁶═CR¹⁶ or —C≡C—;

R¹⁶ is H or optionally substituted alkyl (such as C₁-C₈ alkyl or C₁-C₆alkyl; e.g., methyl, ethyl, or iso-propyl); and

A represents optionally substituted aryl or optionally substitutedheteroaryl.

In certain embodiments, the compound of Formula (III) can be representedby Formula (IIIa) or (IIIb):

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, W is —C≡C—.In other embodiments, W is —CR¹⁶═CR¹⁶ in cis- or trans-formation.

In certain embodiments of any of the foregoing or following, A is aryl,optionally substituted with one or more substituents independentlyselected from the group consisting of —OH, halogen, optionallysubstituted alkyl (such as C₁-C₉ alkyl or C₁-C₆ alkyl; e.g., methyl,ethyl, or iso-propyl), optionally substituted haloalkyl (such as C₁-C₈haloalkyl or C₁-C₆ haloalkyl; e.g., trifluoromethyl), —OR^(a),—OC(O)R^(b), —C(O)NR^(a)R^(b), and —NR^(a)R^(b), and wherein R^(a) andR^(b), independently at each occurrence, are H or optionally substitutedalkyl. In certain such embodiments, A is phenyl, optionally substitutedwith one or more substituents independently selected from the groupconsisting of —CH₂OH, —OH, —CF₃, —COOH, —F, —CH₂NH₂, —CONH₂, and —NH₂.

In certain embodiments of any of the foregoing or following, A isheteroaryl, optionally substituted with one or more substituentsindependently selected from the group consisting of —OH, halogen,optionally substituted alkyl (such as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g.,methyl, ethyl, or iso-propyl), optionally substituted haloalkyl (such asC₁-C₈ haloalkyl or C₁-C₆ haloalkyl; e.g., trifluoromethyl), —OR^(a),—OC(O)R^(b), —C(O)NR^(a)R^(b), and —NR^(a)R^(b), and wherein Re andR^(b), independently at each occurrence, are H or optionally substitutedalkyl (such as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, oriso-propyl).

In certain such embodiments, A is an optionally substituted heteroarylcontaining 1-4 N atoms.

In some embodiments of any of the foregoing or following, A is anoptionally substituted tetrazolyl or optionally substituted triazolyl.

In certain embodiments of any of the foregoing or following, A ispyridinyl, optionally substituted with one or more substituentsindependently selected from the group consisting of —H, —CH₂OOH, —OH,—CF₃, —COOH, —F, —CH₂NH₂, —CONH₂, and —NH₂.

In certain embodiments of any of the foregoing or following, R² isnitro, —F, —Cl, —OCH₃, CCl₃, or —CF₃; R³ is selected from the groupconsisting of isopropyl, cyclopropyl, and cyclobutyl; R⁴ is methyl orisopropyl; and R⁵, R⁶ and R⁷ are each independently selected from thegroup consisting of —H, alkyl (such as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g.,methyl, ethyl, or iso-propyl), phenyl, and —COCH₃.

In certain embodiments of any of the foregoing or following, R² is —CF₃;R³ is iso-propyl; R⁴ is methyl; and R⁵, R⁶, and R⁷ are H.

In certain embodiments, this disclosure provides a compound of Formula(II′):

or a pharmaceutically acceptable salt thereof, wherein:

-   R² and R³ are each independently selected for each occurrence from    the group consisting of —H, halogen (such as F, Br, or Cl),    hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,    —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,    —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl (such    as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted alkenyl, substituted or unsubstituted    alkynyl, substituted or unsubstituted cycloalkyl (such as C₃-C₇    cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted or    unsubstituted heterocyclyl, substituted or unsubstituted aryl,    substituted or unsubstituted heteroaryl, substituted or    unsubstituted arylalkyl, substituted or unsubstituted    heteroarylalkyl, substituted or unsubstituted haloalkyl (such as    C₁-C₈ haloalkyl or C₁-C₆ haloalkyl; e.g., trifluoromethyl), and    substituted or unsubstituted haloalkoxy (such as C₁-C₈ haloalkoxy or    C₁-C₆ haloalkoxy);-   R⁴ is selected from the group consisting of —H, substituted or    unsubstituted alkyl (such as C₁-C₈ alkyl; e.g., methyl, ethyl, or    iso-propyl), substituted or unsubstituted alkenyl, substituted or    unsubstituted alkynyl, substituted or unsubstituted cycloalkyl (such    as C₃-C₇ cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted    or unsubstituted aryl, substituted or unsubstituted heteroaryl,    substituted or unsubstituted arylalkyl, and substituted or    unsubstituted heteroarylalkyl;-   R⁵, R⁶ and R⁷ are each independently selected from the group    consisting of —H, —C(O)R¹¹, substituted or unsubstituted alkyl (such    as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted alkenyl, substituted or unsubstituted    alkynyl, substituted or unsubstituted cycloalkyl (such as C₃-C₇    cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl,    substituted or unsubstituted arylalkyl, and substituted or    unsubstituted heteroarylalkyl; or R⁵ is selected from any of the    foregoing and R⁶ and R⁷ taken together with the nitrogen atom to    which they are attached form a substituted or unsubstituted 3-6    membered ring;-   each occurrence of R¹¹ is independently selected from the group    consisting of substituted or unsubstituted alkyl (such as C₁-C₈    alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted alkenyl, substituted or unsubstituted    alkynyl, substituted or unsubstituted cycloalkyl (such as C₃-C₇    cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted or    unsubstituted heterocyclyl, substituted or unsubstituted aryl, and    substituted or unsubstituted heteroaryl; and-   each occurrence of R¹⁰ and R¹² is independently selected from the    group consisting of —H, substituted or unsubstituted alkyl (such as    C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted alkenyl, substituted or unsubstituted    alkynyl, substituted or unsubstituted cycloalkyl (such as C₃-C₇    cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted or    unsubstituted heterocyclyl, substituted or unsubstituted aryl, and    substituted or unsubstituted heteroaryl.

W represents —CR¹⁶═CR¹⁶— or —C≡C—;

R¹⁶ is H or optionally substituted alkyl (such as C₁-C₈ alkyl or C₁-C₆alkyl; e.g., methyl, ethyl, or iso-propyl); and

L is selected from the group consisting of —H, substituted orunsubstituted alkyl (such as C₁-C₁₂ alkyl or C₁-C₈ alkyl; e.g., methyl,ethyl, iso-propyl, n-butyl, or n-proypl), substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl; e.g., cyclopropyl orcyclobutyl), substituted or unsubstituted heterocyclyl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl.

In certain embodiments, the compound of Formula (II′) can be representedby Formula (IIa′) or (IIb′):

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, W is—CR¹⁶—CR⁶ in a cis-formation. In certain embodiments, W is —CR¹⁶═CR⁶ ina trans-formation. In some embodiments, W is —C≡C—.

In certain embodiments of any of the foregoing or following, L is alkyl,optionally substituted with one or more substituents selected from thegroup consisting of halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted alkyl (such as C₁-C₁₂ alkyl or C₁-C₈ alkyl; e.g., methyl,ethyl, iso-propyl, or n-butyl), substituted or unsubstituted cycloalkyl(such as C₃-C₇ cycloalkyl; e.g., cyclopropyl or cyclobutyl), substitutedor unsubstituted heterocyclyl ring, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl;e.g., trifluoromethyl), and substituted or unsubstituted haloalkoxy(such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy);

In certain embodiments of any of the foregoing or following, L is alkyloptionally substituted with one or more substituents independentlyselected from the group consisting of hydrogen, —OH, halogen, optionallysubstituted alkyl (such as C₁-C₁₂ alkyl or C₁-C₈ alkyl; e.g., methyl,ethyl, iso-propyl, n-butyl), substituted or unsubstituted heteroaryl,optionally substituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆haloalkyl; e.g., trifluoromethyl), —OR¹¹, —OC(O)R¹², —C(O)OR¹²,—C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹²;

In certain embodiments of any of the foregoing or following, L iscycloalkyl or heterocyclyl ring, optionally substituted with one or moresubstituents selected from the group consisting of halogen, hydroxyl,nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹²,—C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹²substituted or unsubstituted alkyl, and oxo.

In certain embodiments of any of the foregoing or following, L is a fourto seven membered heterocyclyl ring comprising one to three heteroatomsselected from the group consisting of NR¹⁰, O, S, SO, or SO₂.

In certain embodiments of any of the foregoing or following, L is H.

In certain embodiments of any of the foregoing or following, thisdisclosure provides a compound of Formula (III′):

or a pharmaceutically acceptable salt thereof, wherein:

-   R² and R³ are each independently selected for each occurrence from    the group consisting of —H, halogen (such as F, Br, or Cl),    hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,    —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,    —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl (such    as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl;    e.g., cyclopropyl or cyclobutyl), substituted or unsubstituted    heterocyclyl, substituted or unsubstituted aryl, substituted or    unsubstituted heteroaryl, substituted or unsubstituted arylalkyl,    substituted or unsubstituted heteroarylalkyl, substituted or    unsubstituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl;    e.g., trifluoromethyl), and substituted or unsubstituted haloalkoxy    (such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy);-   R⁴ is selected from the group consisting of —H, substituted or    unsubstituted alkyl (such as C₁-C₈ alkyl; e.g., methyl, ethyl, or    iso-propyl), substituted or unsubstituted cycloalkyl (such as C₃-C₇    cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted or    unsubstituted aryl, substituted or unsubstituted heteroaryl,    substituted or unsubstituted arylalkyl, and substituted or    unsubstituted heteroarylalkyl;-   R⁵, R⁶ and R⁷ are each independently selected from the group    consisting of —H, —C(O)R¹¹, substituted or unsubstituted alkyl (such    as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl;    e.g., cyclopropyl or cyclobutyl), substituted or unsubstituted aryl,    substituted or unsubstituted heteroaryl, substituted or    unsubstituted arylalkyl, and substituted or unsubstituted    heteroarylalkyl; or R⁵ is selected from any of the foregoing and R⁶    and R⁷ taken together with the nitrogen atom to which they are    attached form a substituted or unsubstituted 3-6 membered ring;-   each occurrence of R¹¹ is independently selected from the group    consisting of substituted or unsubstituted alkyl (such as C₁-C₈    alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted alkenyl, substituted or unsubstituted    alkynyl, substituted or unsubstituted cycloalkyl (such as C₃-C₇    cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted or    unsubstituted heterocyclyl, substituted or unsubstituted aryl, and    substituted or unsubstituted heteroaryl; and-   each occurrence of R¹⁰ and R¹² is independently selected from the    group consisting of —H, substituted or unsubstituted alkyl (such as    C₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),    substituted or unsubstituted alkenyl, substituted or unsubstituted    alkynyl, substituted or unsubstituted cycloalkyl (such as C₃-C₇    cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted or    unsubstituted heterocyclyl, substituted or unsubstituted aryl, and    substituted or unsubstituted heteroaryl.

W represents —CR¹⁶═CR¹⁶ or —C≡C—;

R¹⁶ is H or optionally substituted alkyl (such as C₁-C₈ alkyl or C₁-C₆alkyl; e.g., methyl, ethyl, or iso-propyl); and

A represents optionally substituted aryl or optionally substitutedheteroaryl.

In certain embodiments, the compound of Formula (III) can be representedby Formula (IIIa′) or (IIIb′):

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, W is —C≡C—.In some embodiments, W is —CR¹⁶═CR¹⁶ in a cis-formation. In someembodiments, W is in a trans-formation.

In certain embodiments of any of the foregoing or following, A is aryl,optionally substituted with one or more substituents independentlyselected from the group consisting of halogen (such as F, Br, or Cl),hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹,—NS(O)₂R¹², substituted or unsubstituted alkyl (such as C₁-C₈ alkyl orC₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl), substituted orunsubstituted alkenyl, substituted or unsubstituted alkynyl, substitutedor unsubstituted cycloalkyl (such as C₃-C₇ cycloalkyl; e.g., cyclopropylor cyclobutyl), substituted or unsubstituted heterocyclyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted haloalkyl (such as C₁-C₈haloalkyl or C₁-C₆ haloalkyl; e.g., trifluoromethyl), and substituted orunsubstituted haloalkoxy (such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy);

In certain such embodiments, A is phenyl, optionally substituted withone or more substituents independently selected from the groupconsisting of —CH₂OH, —OH, —CF₃, —COOH, —F, —CH₂NH₂, —CONH₂, and —NH₂.

In certain embodiments of any of the foregoing or following, A isheteroaryl, optionally substituted with one or more substituentsindependently selected from the group consisting of halogen (such as F,Br, or Cl), hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹²,—OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl (such asC₁-C₈ alkyl or C₁-C₆ alkyl; e.g., methyl, ethyl, or iso-propyl),substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl (such as C₃-C₇cycloalkyl; e.g., cyclopropyl or cyclobutyl), substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted haloalkyl (such as C₁-C₈ haloalkyl or C₁-C₆ haloalkyl;e.g., trifluoromethyl), and substituted or unsubstituted haloalkoxy(such as C₁-C₈ haloalkoxy or C₁-C₆ haloalkoxy);

In certain embodiments of any of the foregoing or following, A isheteroaryl, optionally substituted with one or more substituentsindependently selected from the group consisting of —OH, halogen,optionally substituted alkyl (such as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g.,methyl, ethyl, or iso-propyl), optionally substituted haloalkyl (such asC₁-C₈ haloalkyl or C₁-C₆ haloalkyl; e.g., trifluoromethyl), —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹².

In certain such embodiments, A is an optionally substituted heteroarylcontaining 1-4 N atoms.

In some embodiments of any of the foregoing or following, A is anoptionally substituted tetrazolyl or optionally substituted triazolyl.

In certain embodiments of any of the foregoing or following, A ispyridinyl, optionally substituted with one or more substituentsindependently selected from the group consisting of —H, —CH₂OH, —OH,—CF₃, —COOH, —F, —CH₂NH₂, —CONH₂, and —NH₂.

In certain embodiments of any of the foregoing or following, R² isnitro, —F, —Cl, —OCH₃, CCl₃, or —CF₃; R³ is selected from the groupconsisting of isopropyl, cyclopropyl, and cyclobutyl; R⁴ is methyl orisopropyl; and R⁵, R⁶ and R⁷ are each independently selected from thegroup consisting of —H, alkyl (such as C₁-C₈ alkyl or C₁-C₆ alkyl; e.g.,methyl, ethyl, or iso-propyl), phenyl, and —COCH₃.

In certain embodiments of any of the foregoing or following, R² is —CF₃;R³ is iso-propyl; R⁴ is methyl; and R⁵, R⁶, and R⁷ are H.

In certain embodiments of any of the foregoing or following, thisdisclosure provides a compound selected from any compounds describedherein, such as the group consisting of compounds as shown in Table 1 or3, and pharmaceutically acceptable salts thereof. Such compounds may beused in any of the methods described herein, such as for the treatmentof a lymphoma, such as a B cell lymphoma, or to inhibit growth,proliferation and/or survival of a lymphoma cell.

In certain embodiments of any of the foregoing or following, thisdisclosure provides a pharmaceutical composition comprising (a) acompound described in this disclosure; and (b) one or morepharmaceutically acceptable carriers and/or excipients.

In certain embodiments of any of the foregoing or following, thecompound of disclosure is selected from Table 1, and pharmaceuticalacceptable salts thereof.

TABLE 1 1,4-dihydropyrano[2,3-c]pyrazole derivatives as SHMT inhibitorsCompound # Chemical Structure  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

Certain compounds of the disclosure (e.g., Compounds of Formula (I),(II), and (II′) and their corresponding inhibitory activity values areshown in Table 2 below. The assay used to evaluate activity is describedin the Examples.

TABLE 2 HCT SHMT2 SHMT1 Cell % LogP IC50 IC50 IC50 formate Compound #Structure Value (μM) (μM) (μM) rescue  4

3.56 0.00017 — 15.10 93.35 68

2.37 0.0004 0.0017 100.00 106.02  1 (enantiomeric mixture)

3.91 0.00065 — — —  1 (enatiomerically active peak)

3.91 0.00079 — 1.36 94.96 69

2.49 0.0008 — 100.00 96.41 70

2.49 0.001 — 100.00 94.89 11

3.22 0.0014 0.0004 5.98 96.98 71

2.89 0.0017 0.0024 4.96 97.81 72

3.38 0.002 0.004 200.00 98.35  2

3.46 0.004 0.002 6.90 100.58 73

4.17 0.0075 — 11.10 97.33  8

2.72 0.043 0.008 2.04 80.65 74

0.019 0.017

D. More Compounds

In one aspect, the disclosure provides compounds represented by generalFormula IV:

wherein:

R⁰, R¹ and R² are each independently selected from —H, halogen,hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—OC(O)R¹², —C(O)OR¹², —C(O)R¹⁰R¹², —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted C₁-C₆ haloalkyl, or substituted or unsubstituted C₁-C₆haloalkoxy;

R³ is selected from —H, halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy;

R⁴ is selected from H, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, or substituted or unsubstitutedheteroarylalkyl; R, R⁶ and R⁷ are each independently selected from —H,—C(O)R¹¹, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, substituted or unsubstituted arylalkyl, orsubstituted or unsubstituted heteroarylalkyl, or R⁵ is selected from anyof the foregoing and R⁶ and R⁷ taken together with the nitrogen atom towhich they are attached form a substituted or unsubstituted 3-6 memberedring; with the proviso that the occurrences of R⁵, R⁶ and R⁷ are not allH simultaneously;

each occurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl; and each occurrence ofR¹⁰ and R¹² is each independently selected from —H, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl, or a pharmaceuticallyacceptable salt thereof.

In certain embodiments, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, are represented by Formula(IVa) (wherein the R groups are as described above for Formula (IV):

In certain embodiments, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, are represented by Formula(IVb) (wherein the R groups are as described above for Formula (IV)):

In certain embodiments of any of the foregoing or following, R⁰, R¹ andR² are each independently selected from —H, halogen, hydroxyl, nitro,nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy.

In certain embodiments of any of the foregoing or following, R⁰ isselected from hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹²,—NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹². In other embodiments, R⁰ isselected from —H, halogen, substituted or unsubstituted C₁-C₆ haloalkyl,substituted or unsubstituted C₁-C₆ haloalkoxy, or substituted orunsubstituted C₁-C₆ alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²(and, optionally R⁰) are each independently selected from —H, halogen,hydroxyl, nitro, nitrile, —OR¹¹, substituted or unsubstituted C₁-C₆alkyl, substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy. In other embodiments, R¹ and R² (and,optionally R⁰) are each independently selected from —H, halogen,substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl.

In certain embodiments of any of the foregoing or following, R¹ and R²(and, optionally R⁰) are each independently selected from —H, methoxy,fluoro, chloro, bromo, hydroxyl, nitro, nitrile, methyl,trifluoromethyl, or trifluoromethoxy. In other embodiments, R⁰ isselected from —H, halogen, substituted or unsubstituted C₁-C₆ haloalkyl,substituted or unsubstituted C₁-C₆ haloalkoxy, or substituted orunsubstituted C₁-C₆ alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²(and, optionally R⁰) are each independently selected from —H, methoxy,chloro, nitro, nitrile, or trifluoromethyl. In other embodiments, R⁰ isselected from —H, halogen, substituted or unsubstituted C₁-C₆ haloalkyl,substituted or unsubstituted C₁-C₆ haloalkoxy, or substituted orunsubstituted C₁-C₆ alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²(and, optionally, R⁰) are each trifluoromethyl. In other embodiments, R⁰is selected from —H, halogen, substituted or unsubstituted C₁-C₆haloalkyl, substituted or unsubstituted C₁-C₆ haloalkoxy, or substitutedor unsubstituted C₁-C₆ alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R³ isselected from —H, halogen, hydroxyl, nitro, nitrile, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected from substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected from substituted or unsubstituted C₁-C₆ alkyl, or substitutedor unsubstituted cycloalkyl.

In certain embodiments of any of the foregoing or following, R³ isselected from methyl, ethyl, propyl, isopropyl, cyclopropyl, orcyclobutyl. In certain embodiments, any of the foregoing may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R³ isselected from isopropyl, cyclopropyl, or cyclobutyl. In certainembodiments, any of the foregoing may be optionally substituted.

In certain embodiments of any of the foregoing or following, R³ iscyclobutyl. In certain embodiments, a cyclobutyl may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, R⁴ isselected from —H, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or substituted or unsubstitutedarylalkyl.

In certain embodiments of any of the foregoing or following, R⁴ isselected from —H, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted arylalkyl.

In certain embodiments of any of the foregoing or following, R⁴ isselected from methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, phenyl or benzyl. In certain embodiments, anyof the foregoing may be optionally substituted.

In certain embodiments of any of the foregoing or following, R⁴ ismethyl or isopropyl. In certain embodiments, any of the foregoing may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R⁴ ismethyl. In certain embodiments, methyl may be optionally substituted.

In certain embodiments of any of the foregoing or following, R⁵, R⁶ andR⁷ are each independently selected from —H, —C(O)R¹¹, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedarylalkyl, or R⁵ is selected from any of the foregoing and R⁶ and R⁷taken together with the nitrogen atom to which they are attached form asubstituted or unsubstituted 3-6 membered ring.

In certain embodiments of any of the foregoing or following, R⁵, R⁶ andR⁷ are each independently selected from —H, —C(O)R¹¹, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedarylalkyl.

In certain embodiments of any of the foregoing or following, R⁵, R⁶ andR⁷ are each independently selected from —H, methyl, ethyl, isopropyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, benzyl, or—COCH₃. In certain embodiments, any of the foregoing, except —H, may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R⁵, R⁶ andR⁷ are each independently selected from —H, methyl, phenyl, or —COCH₃.In certain embodiments, any of the foregoing, except —H, may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R⁵ and R⁶are each independently selected from —H, methyl or phenyl. In certainembodiments, any of the foregoing, except —H, may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, R⁷ is —H.

In certain embodiments of any of the foregoing or following, R⁶ and R⁷taken together with the nitrogen atom to which they are attached form asubstituted or unsubstituted 3-6 membered ring. In certain embodimentsthe 3-6 membered ring is a monocyclic ring. In certain embodiments, the3-6 membered ring may be saturated or unsaturated (e.g., contain atleast one double bond). In certain embodiments, the 3-6 membered ringmay contain one or two additional heteroatoms, other than the nitrogenatom to which R⁶ and R⁷ are attached.

In certain embodiments of any of the foregoing or following, R⁵ isselected from —H, methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, phenyl, benzyl, or —COCH₃, and R⁶ and R⁷ takentogether with the nitrogen atom to which they are attached form asubstituted or unsubstituted ring selected from:

In certain embodiments of any of the foregoing or following, R⁵ isselected from —H, methyl, phenyl, or —COCH₃, and R⁶ and R⁷ takentogether with the nitrogen atom to which they are attached form asubstituted or unsubstituted ring selected from:

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹⁰ is independently selected from substituted orunsubstituted C₁-C₆ alkyl or substituted or unsubstituted cycloalkyl. Incertain embodiments, there is no occurrence of R¹¹.

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹⁰ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted cycloalkyl, such as from —H, or substituted orunsubstituted C₁-C₆ alkyl. In certain embodiments, each occurrence ofR¹⁰ and R¹² is —H. In certain embodiments, there is no occurrence of R⁰and/or R¹².

In certain embodiments of any of the foregoing or following, R⁰ isselected from —H, halogen, hydroxyl, nitro, nitrile, —S(O)₂R¹¹,—S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹,—NS(O)₂R¹², substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstituted C₁-C₆haloalkyl, or substituted or unsubstituted C₁-C₆ haloalkoxy;

R¹ and R² are each independently selected from —H, methoxy, fluoro,chloro, bromo, hydroxyl, nitro, nitrile, methyl, or trifluoromethyl;

R³ is selected from methyl, ethyl, propyl, isopropyl, cyclopropyl, orcyclobutyl;

R⁴ is selected from methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, phenyl or benzyl; and

R⁵, R⁶ and R⁷ are each independently selected from —H, methyl, ethyl,isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl,benzyl, or —COCH₃.

In certain embodiments of any of the foregoing or following, R⁰ isselected from —H, hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹²;

R¹ and R² are each independently selected from —H, methoxy, chloro,nitro, nitrile, or trifluoromethyl;

R³ is selected from isopropyl, cyclopropyl, or cyclobutyl;

R⁴ is methyl or isopropyl; and

R⁵, R⁶ and R⁷ are each independently selected from —H, methyl, phenyl,or —COCH₃.

In certain embodiments of any of the foregoing or following, R⁰ isselected from —H, hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹²;

R¹ and R² are each —CF₃;

R³ is cyclobutyl;

R⁴ is methyl;

R⁵ and R⁶ are each independently selected from H, methyl or phenyl; and

R⁷ is H.

In certain embodiments, the compound is selected from the groupconsisting of:

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, thedisclosure provides a pharmaceutically acceptable salt thereof or anenantiomer thereof.

In certain embodiments of any of the foregoing or following, the R⁰, R¹and R² are not all simultaneously —H. In other embodiments, R⁰ is —H andthe ring to which it is attached, is substituted with a singlesubstituent (other than —H) at one of R¹ or R². In certain embodiments,R⁰ is —H, and R¹ and R² are not —H. In other embodiments, R¹ is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R². In certain embodiments,R¹ is —H, and R⁰ and R² are not —H. In other embodiments, R² is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R¹. In certain embodiments,R² is —H, and R⁰ and R¹ are not —H. In other embodiments, R⁰, R¹ and R²are not —H.

In one aspect, the disclosure provides compounds represented by generalFormula (V):

wherein:

Z is N or CR⁴;

X is O, S, CH₂, or NR⁶;

R⁰, R¹ and R² are each independently selected from —H, halogen,hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹,—NS(O)₂R¹², substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstituted C₁-C₆haloalkyl, or substituted or unsubstituted C₁-C₆ haloalkoxy;

R³ is selected from —H, halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy;

R⁴ is selected from —H, —NR¹⁰R¹², —C(O)NR¹⁰R¹², —N(R²)C(O)R¹¹, nitrile,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl, orsubstituted or unsubstituted heteroarylalkyl;

R⁵ is selected from —H, —NR¹⁰R¹², —C(O)NR¹⁰R¹², —N(R²)C(O)R¹¹, nitrile,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl, orsubstituted or unsubstituted heteroarylalkyl, or R⁵ and R⁴ takentogether with the respective carbon atom to which they are attached forma substituted or unsubstituted 4-12 membered ring;

R⁶ is selected from H, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl, orsubstituted or unsubstituted heteroarylalkyl;

R⁷ and Re are each independently selected from —H, —NR¹⁰R¹²,—C(O)NR¹⁰R¹², —N(R¹²)C(O)R¹¹, nitrile, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted arylalkyl, or substituted or unsubstitutedheteroarylalkyl;

each occurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl; and

each occurrence of R¹⁰ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, or apharmaceutically acceptable salt thereof.

In certain embodiments, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, are represented by Formula(Va) (wherein the R groups are as described above for Formula (V)):

In certain embodiments, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, are represented Formula (Vb)(wherein the R groups are as described above for Formula (V)):

In certain embodiments of any of the foregoing or following, R⁰, R¹ andR² are each independently selected from —H, halogen, hydroxyl, nitro,nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy.

In certain embodiments of any of the foregoing or following, R⁰ isselected from hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹²,—NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹². In other embodiments, R⁰ isselected from —H, halogen, substituted or unsubstituted C₁-C₆ haloalkyl,substituted or unsubstituted C₁-C₆ haloalkoxy, or substituted orunsubstituted C₁-C₆ alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each independently selected from —H, halogen, hydroxyl, nitro,nitrile, —OR¹¹, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy. In other embodiments, R⁰ is selected from —H, halogen,substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each independently selected from —H, methoxy, fluoro, chloro, bromo,hydroxyl, nitro, nitrile, methyl, trifluoromethyl, or trifluoromethoxy.In other embodiments, R⁰ is selected from —H, halogen, substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy, or substituted or unsubstituted C₁-C₆ alkyl. In otherembodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each trifluoromethyl. In other embodiments, R⁰ is selected from —H,halogen, substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R³ isselected from —H, halogen, hydroxyl, nitro, nitrile, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected from methyl, ethyl, propyl, isopropyl, cyclopropyl, orcyclobutyl. In certain embodiments, any of the foregoing may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R³ isisopropyl or cyclopropyl. In certain embodiments, any of the foregoingmay be optionally substituted.

In certain embodiments of any of the foregoing or following, R⁴ and R⁵are each independently selected from —H, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, orsubstituted or unsubstituted arylalkyl.

In certain embodiments of any of the foregoing or following, R⁴ and R⁵are each independently selected from —H, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted arylalkyl.

In certain embodiments of any of the foregoing or following, R⁴ and R⁵are each independently selected from —H, methyl, ethyl, propyl,isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl orbenzyl. In certain embodiments, any of the foregoing, except —H, may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R⁴ and R⁵are independently —H or methyl. In certain embodiments, methyl may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R⁵ and R⁴taken together with the respective carbon atom to which they areattached form a 4-12 membered ring selected from substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

In certain embodiments of any of the foregoing or following, R⁵ and R⁴taken together with the respective carbon atom to which they areattached form a substituted or unsubstituted 4-12 membered ringcontaining 0-4 heteroatoms (0, 1, 2, 3 or 4) independently selected fromN, O, or S.

In certain embodiments of any of the foregoing or following, the 4-12membered ring is a monocyclic ring. In certain embodiments of any of theforegoing or following, the 4-12 membered ring is a polycyclic ring. Incertain embodiments of any of the foregoing or following, the 4-12membered ring is a bicyclic ring. In certain embodiments of any of theforegoing or following, when the 4-12 membered ring is a polycyclicring, each ring is independently selected from saturated or unsaturated,and each ring may independently contain one or more heteroatoms (e.g.,for a total of 1, 2, 3, 4 or 4 heteroatoms).

In certain embodiments of any of the foregoing or following, R⁵ and R⁴taken together with the respective carbon atom to which they areattached form a phenyl ring. In certain embodiments, the phenyl ring maybe optionally substituted.

In certain embodiments of any of the foregoing or following, R⁶ isselected from H, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or substituted or unsubstitutedarylalkyl.

In certain embodiments of any of the foregoing or following, R⁶ isselected from —H, methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, phenyl, pyridyl, or benzyl. In certainembodiments, any of the foregoing may be optionally substituted.

In certain embodiments of any of the foregoing or following, R⁶ is —H.

In certain embodiments of any of the foregoing or following, R⁷ and R⁸are each independently selected from —H, —NR¹⁰R¹², —C(O)NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, nitrile, methyl, ethyl, isopropyl, cyclopropryl,cyclobutyl, cyclopentyl, cyclohexyl, phenyl, or benzyl.

In certain embodiments of any of the foregoing or following, R⁷ isselected from —H, —NH₂, methyl, or phenyl. In certain embodiments, anyof the foregoing except —H, may be optionally substituted.

In certain embodiments of any of the foregoing or following, R⁸ isselected from —H, nitrile, or —C(O)NH₂. In certain embodiments, —C(O)NH₂may be optionally substituted.

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl or substituted or unsubstituted cycloalkyl. Incertain embodiments, there is no occurrence of R¹¹.

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹⁰ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted cycloalkyl, such as from —H, or substituted orunsubstituted C₁-C₆ alkyl. In certain embodiments, each occurrence ofR¹⁰ and R¹² is —H. In certain embodiments, there is no occurrence of R⁰and/or R¹².

In certain embodiments of any of the foregoing or following, thedisclosure provides a pharmaceutically acceptable salt thereof or anenantiomer thereof.

In certain embodiments of any of the foregoing or following, the R⁰, R¹and R² are not all simultaneously —H. In other embodiments, R⁰ is —H andthe ring to which it is attached, is substituted with a singlesubstituent (other than —H) at one of R¹ or R². In certain embodiments,R⁰ is —H, and R¹ and R² are not —H. In other embodiments, R¹ is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R². In certain embodiments,R¹ is —H, and R⁰ and R² are not —H. In other embodiments, R² is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R¹. In certain embodiments,R² is —H, and R⁰ and R¹ are not —H. In other embodiments, R⁰, R¹ and R²are not —H.

In certain embodiments, when Z is N, and R⁸ is H, R⁷ cannot besubstituted or unsubstituted aryl.

In certain embodiments, the compound of Formulae (V), (Va), or (Vb) isnot

In certain embodiments, when R⁴ and R⁵ taken together with therespective carbon atoms to which they are attached for a substitutedaryl ring, R⁸ is not —H, —OH, or —CN.

In certain embodiments, the compound of Formulae (V), (Va), or (Vb) is:

or a pharmaceutically acceptable salt thereof.

In one aspect, the disclosure provides compounds represented by generalby Formula (VI):

wherein:

R⁰, R¹ and R² are each independently selected from —H, halogen,hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹,—NS(O)₂R¹², substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstituted C₁-C₆haloalkyl, or substituted or unsubstituted C₁-C₆ haloalkoxy;

R³ is selected from —H, halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy;

R⁵ is selected from —H, —NR¹⁰R¹², —C(O)NR¹⁰R¹², —N(R¹²)C(O)R¹¹, nitrile,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl, orsubstituted or unsubstituted heteroarylalkyl;

R⁶ is selected from —H, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl substituted or unsubstituted arylalkyl, orsubstituted or unsubstituted heteroarylalkyl;

R⁷ and R⁸ are each independently selected from —H, —NR¹⁰R¹²,—C(O)NR¹⁰R¹², —N(R¹²)C(O)R¹¹, nitrile, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted arylalkyl, or substituted or unsubstitutedheteroarylalkyl;

each occurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl; and

each occurrence of R¹⁰ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, or apharmaceutically acceptable salt thereof.

In certain embodiments, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, are represented by Formula(VIa) (wherein the R groups are as described above for Formula (VI)):

In certain embodiments, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, are represented by Formula(VIb) (wherein the R groups are as described above for Formula (VI)):

In certain embodiments of any of the foregoing or following, R⁰, R¹ andR² are each independently selected from —H, halogen, hydroxyl, nitro,nitrile, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, or —NS(O)₂R¹², substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedC₁-C₆ haloalkyl, or substituted or unsubstituted C₁-C₆ haloalkoxy.

In certain embodiments of any of the foregoing or following, R⁰ isselected from hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹²,—NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹². In other embodiments, R⁰ isselected from —H, halogen, substituted or unsubstituted C₁-C₆ haloalkyl,substituted or unsubstituted C₁-C₆ haloalkoxy, or substituted orunsubstituted C₁-C₆ alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each independently selected from —H, halogen, hydroxyl, nitro,nitrile, —OR¹¹, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted C₁-C₆ haloalkyl, or substituted or unsubstituted C₁-C₆haloalkoxy. In other embodiments, R⁰ is selected from —H, halogen,substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each independently selected from —H, methoxy, fluoro, chloro, bromo,hydroxyl, nitro, nitrile, methyl, trifluoromethyl, or trifluoromethoxy.In other embodiments, R⁰ is selected from —H, halogen, substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy, or substituted or unsubstituted C₁-C₆ alkyl. In otherembodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each trifluoromethyl. In other embodiments, R⁰ is selected from —H,halogen, substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R³ isselected from —H, halogen, hydroxyl, nitro, nitrile, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected from methyl, ethyl, propyl, isopropyl, cyclopropyl, orcyclobutyl. In certain embodiments, any of the foregoing may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R³ isisopropyl or cyclopropyl. In certain embodiments, any of the foregoingmay be optionally substituted.

In certain embodiments of any of the foregoing or following, R⁵ isselected from —H, methyl, ethyl, propyl, isopropyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, phenyl or benzyl. In certainembodiments, any of the foregoing except —H, may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, R⁵ is —H.

In certain embodiments of any of the foregoing or following, R⁶ isselected from H, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or substituted or unsubstitutedarylalkyl.

In certain embodiments of any of the foregoing or following, R⁶ isselected from —H, methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, phenyl, pyridyl or benzyl. In certainembodiments, any of the foregoing except —H, may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, R⁶ is —H.

In certain embodiments of any of the foregoing or following, R⁷ and R⁸are each independently selected from —H, —NR¹⁰R¹², —C(O)NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, nitrile, methyl, ethyl, isopropyl, cyclopropryl,cyclobutyl, cyclopentyl, cyclohexyl, phenyl, or benzyl.

In certain embodiments of any of the foregoing or following, R⁷ isselected from —NH₂, methyl, or phenyl. In certain embodiments, any ofthe foregoing, may be optionally substituted.

In certain embodiments of any of the foregoing or following, R⁸ isselected from —H, nitrile, or —C(O)NH₂. In certain embodiments, —C(O)NH₂may be optionally substituted.

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl or substituted or unsubstituted cycloalkyl. Incertain embodiments, there is no occurrence of R¹¹.

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹¹ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted cycloalkyl, such as from —H, or substituted orunsubstituted C₁-C₆ alkyl. In certain embodiments, each occurrence ofR¹⁰ and R¹² is —H. In certain embodiments, there is no occurrence of R¹⁰and/or R¹².

In certain embodiments of any of the foregoing or following, R⁰ isselected from —H, halogen, hydroxyl, nitro, nitrile, —S(O)₂R¹¹,—S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or—NS(O)₂R¹², substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted C₁-C₆ haloalkyl, or substituted or unsubstituted C₁-C₆haloalkoxy;

R¹ and R² are each independently selected from —H, methoxy, fluoro,chloro, bromo, hydroxyl, nitro, nitrile, methyl, or trifluoromethyl;

R³ is selected from methyl, ethyl, propyl, isopropyl, cyclopropyl, orcyclobutyl;

R⁵ is selected from —H, methyl, ethyl, propyl or isopropyl;

R⁶ is selected from —H, methyl, ethyl, isopropyl, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, phenyl or benzyl; and R⁷ and R⁸ areeach independently selected from —H, —NH₂, —C(O)NH₂, nitrile, methyl,ethyl, isopropyl, cyclopropryl, cyclobutyl, cyclopentyl, cyclohexyl,phenyl, or benzyl.

In certain embodiments of any of the foregoing or following, R⁰ isselected from —H, hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹²;

R¹ and R² are each trifluoromethyl;

R³ is isopropyl or cyclopropyl;

R⁵ is —H;

R⁶ is —H;

R⁷ is selected from —NH₂, methyl, or phenyl; and

R⁸ is selected from H, nitrile, or —C(O)NH₂.

In certain embodiments, the compound is selected from:

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, thedisclosure provides a pharmaceutically acceptable salt thereof or anenantiomer thereof.

In certain embodiments of any of the foregoing or following, the R⁰, R¹and R² are not all simultaneously —H. In other embodiments, R⁰ is —H andthe ring to which it is attached, is substituted with a singlesubstituent (other than —H) at one of R¹ or R². In certain embodiments,R⁰ is —H, and R¹ and R² are not —H. In other embodiments, R¹ is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R². In certain embodiments,R¹ is —H, and R⁰ and R² are not —H. In other embodiments, R² is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R¹. In certain embodiments,R² is —H, and R⁰ and R¹ are not —H. In other embodiments, R⁰, R¹ and R²are not —H.

In certain embodiments, when Re is H, R⁷ is not a substituted orunsubstituted aryl.

In one aspect, the disclosure provides compounds represented by generalFormula (VII):

wherein:

R⁰, R¹ and R² are each independently selected from —H, halogen,hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹,—NS(O)₂R¹², substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, substituted orunsubstituted heteroarylalkyl, substituted or unsubstituted C₁-C₆haloalkyl, or substituted or unsubstituted C₁-C₆ haloalkoxy;

R³ is selected from —H, halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy;

R⁴ and R⁵ are independently selected from H, —NR¹⁰R¹², —C(O)NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, nitrile, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, or substituted or unsubstitutedheteroarylalkyl, or R⁵ and R⁴ taken together with the respective carbonatom to which they are attached form a substituted or unsubstituted 4-12membered ring;

R⁶ is selected from —H, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl, orsubstituted or unsubstituted heteroarylalkyl;

R⁷ and R⁸ are each independently selected from —H, —NR¹⁰R¹²,—C(O)NR¹⁰R¹², —N(R¹²)C(O)R¹¹, nitrile, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted arylalkyl, or substituted or unsubstitutedheteroarylalkyl;

each occurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl; and

each occurrence of R¹⁰ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, or apharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, thecompounds of the disclosure, or a pharmaceutically acceptable saltthereof, are represented by Formula (VIIa) (wherein the R groups are asdescribed above for Formula (VII)):

In certain embodiments of any of the foregoing or following, thecompounds of the disclosure, or a pharmaceutically acceptable saltthereof, are represented by Formula (VIIb) (wherein the R groups are asdescribed above for Formula (VII)):

In certain embodiments of any of the foregoing or following, R⁰, R¹ andR² are each independently selected from —H, halogen, hydroxyl, nitro,nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy.

In certain embodiments of any of the foregoing or following, R⁰ isselected from hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹²,—NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹². In other embodiments, R⁰ isselected from —H, halogen, substituted or unsubstituted C₁-C₆ haloalkyl,substituted or unsubstituted C₁-C₆ haloalkoxy, or substituted orunsubstituted C₁-C₆ alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each independently selected from —H, hydroxyl, —S(O)₂R¹¹,—S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or—NS(O)₂R¹².

In certain embodiments of any of the foregoing or following, R¹ and R²are each independently selected from —H, methoxy, fluoro, chloro, bromo,hydroxyl, nitro, nitrile, methyl, trifluoromethyl, or trifluoromethoxy.In other embodiments, R⁰ is selected from —H, halogen, substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy, or substituted or unsubstituted C₁-C₆ alkyl. In otherembodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each trifluoromethyl. In other embodiments, R⁰ is selected from —H,halogen, substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R³ isselected from —H, halogen, hydroxyl, nitro, nitrile, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected from methyl, ethyl, propyl, isopropyl, cyclopropyl, orcyclobutyl. In certain embodiments, any of the foregoing, may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R³ isisopropyl. In certain embodiments, isopropyl may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, R⁴ and R⁵are independently selected from H, substituted or unsubstituted C₁-C₆alkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted arylalkyl, or substituted or unsubstitutedheteroarylalkyl.

In certain embodiments of any of the foregoing or following, R⁴ and R⁵are each independently selected from —H, methyl, ethyl, propyl,isopropyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl orbenzyl. In certain embodiments, any of the foregoing except —H, may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R⁴ and R⁵are each independently —H or methyl. In certain embodiments, methyl maybe optionally substituted.

In certain embodiments of any of the foregoing or following, R⁵ and R⁴taken together with the respective carbon atoms to which they areattached form a substituted or unsubstituted 4-12 membered ring.

In certain embodiments of any of the foregoing or following, R⁵ and R⁴taken together with the respective carbon atom to which they areattached form a 4-12 membered ring selected from substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl.

In certain embodiments of any of the foregoing or following, R⁵ and R⁴taken together with the respective carbon atom to which they areattached form a substituted or unsubstituted 4-12 membered ringcontaining 0-4 heteroatoms (0, 1, 2, 3 or 4) independently selected fromN, O, or S.

In certain embodiments of any of the foregoing or following, the 4-12membered ring is a monocyclic ring. In certain embodiments of any of theforegoing or following, the 4-12 membered ring is a polycyclic ring. Incertain embodiments of any of the foregoing or following, the 4-12membered ring is a bicyclic ring. In certain embodiments of any of theforegoing or following, when the 4-12 membered ring is a polycyclicring, each ring is independently selected from saturated or unsaturated,and each ring may independently contain one or more heteroatoms.

In certain embodiments of any of the foregoing or following, R⁵ and R⁴taken together with the respective carbon atom to which they areattached form a phenyl ring. In certain embodiments, the phenyl ring maybe optionally substituted.

In certain embodiments of any of the foregoing or following, R⁶ isselected from H, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or substituted or unsubstitutedarylalkyl.

In certain embodiments of any of the foregoing or following, R⁶ isselected from —H, methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, phenyl, pyridyl, or benzyl. In certainembodiments, any of the foregoing except —H, may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, R⁶ is —H.

In certain embodiments of any of the foregoing or following, R⁷ and R⁸are each independently selected from —H, —NR¹⁰R¹², —C(O)NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, nitrile, methyl, ethyl, isopropyl, cyclopropryl,cyclobutyl, cyclopentyl, cyclohexyl, phenyl, or benzyl.

In certain embodiments of any of the foregoing or following, R⁷ isselected from —H, —NH₂, methyl, or phenyl. In certain embodiments, anyof the foregoing except —H, may be optionally substituted.

In certain embodiments of any of the foregoing or following, R⁸ isselected from —H, nitrile, or —C(O)NH₂. In certain embodiments, —C(O)NH₂may be optionally substituted.

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl or substituted or unsubstituted cycloalkyl. Incertain embodiments, there is no occurrence of R¹¹.

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹⁰ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted cycloalkyl, such as from —H, or substituted orunsubstituted C₁-C₆ alkyl. In certain embodiments, each occurrence ofR¹⁰ and R¹² is —H. In certain embodiments, there is no occurrence of R⁰and/or R¹².

In certain embodiments, the compound is selected from:

or a pharmaceutically acceptable salt thereof.

In certain embodiments, the compound of Formula (VII), (VIIa), or (VIIb)is:

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, the R⁰, R¹and R² are not all simultaneously —H. In other embodiments, R⁰ is —H andthe ring to which it is attached, is substituted with a singlesubstituent (other than —H) at one of R¹ or R². In certain embodiments,R⁰ is —H, and R¹ and R² are not —H. In other embodiments, R¹ is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R². In certain embodiments,R¹ is —H, and R⁰ and R² are not —H. In other embodiments, R² is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R¹. In certain embodiments,R² is —H, and R⁰ and R¹ are not —H. In other embodiments, R⁰, R¹ and R²are not —H.

In certain embodiments, the compound of Formula (VII), (VIIa), or (VIIb)is not

In one aspect, the disclosure provides compounds represented by generalFormula (VIII):

wherein:

A, D, G and K are each independently N or CR¹⁵, provided that no morethan two of A, D, G, and K are N simultaneously;

R⁰, R¹ and R² are each independently selected from —H, halogen,hydroxyl, nitro, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹²,—C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹²substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl substitutedor unsubstituted arylalkyl, substituted or unsubstitutedheteroarylalkyl, substituted or unsubstituted C₁-C₆ haloalkyl, orsubstituted or unsubstituted C₁-C₆ haloalkoxy;

R³ is selected from —H, halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy;

R⁷ and R⁸ are each independently selected from —H, —NR¹⁰R¹²,—C(O)NR¹⁰R¹², —N(R¹²)C(O)R¹¹, nitrile, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted arylalkyl, or substituted or unsubstitutedheteroarylalkyl;

each occurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl;

each occurrence of R¹⁰ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; and eachoccurrence of R¹⁵ is independently selected from —H, halogen, hydroxyl,nitro, nitrile, substituted or unsubstituted C₁-C₆ alkoxy, substitutedor unsubstituted C₁-C₆ alkyl, or substituted or unsubstituted C₁-C₆haloalkyl, substituted or unsubstituted C₁-C₆ haloalkoxy, or apharmaceutically acceptable salt thereof.

In certain embodiments, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, are represented by Formula(VIIIa) (wherein the R groups are as described above for Formula(VIII)):

In certain embodiments, the compounds of the disclosure, or apharmaceutically acceptable salt thereof, are represented by Formula(VIIIb) (wherein the R groups are as described above for Formula(VIII)):

In certain embodiments of any of the foregoing or following, R⁰, R¹, andR², are each independently selected from —H, halogen, hydroxyl, nitro,nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy.

In certain embodiments of any of the foregoing or following, R⁰ isselected from —H, hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹². In otherembodiments, R⁰ is selected from —H, halogen, substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy, or substituted or unsubstituted C₁-C₆ alkyl. In otherembodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each independently selected from —H, halogen, hydroxyl, nitro,nitrile, —OR₁₁, substituted or unsubstituted C₁-C₆ alkyl, or substitutedor unsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy. In other embodiments, R⁰ is selected from —H, halogen,substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each independently selected from —H, methoxy, fluoro, chloro, bromo,hydroxyl, nitro, nitrile, methyl, or trifluoromethyl, ortrifluoromethoxy. In other embodiments, R⁰ is selected from —H, halogen,substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R¹ and R²are each trifluoromethyl. In other embodiments, R⁰ is selected from —H,halogen, substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R³ isselected from —H, halogen, hydroxyl, nitro, nitrile, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected from methyl, ethyl, propyl, isopropyl, cyclopropyl, orcyclobutyl. In certain embodiments, any of the foregoing, may beoptionally substituted.

In certain embodiments, in of any of the foregoing or following, R³ isisopropyl. In certain embodiments, isopropyl may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, R⁷ and R⁸are each independently selected from —H, —NR¹⁰R¹², —C(O)NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, nitrile, methyl, ethyl, isopropyl, cyclopropryl,cyclobutyl, cyclopentyl, cyclohexyl, phenyl, or benzyl. In certainembodiments, any of the foregoing except —H, may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, R⁷ ismethyl or phenyl. In certain embodiments, any of the foregoing may beoptionally substituted.

In certain embodiments of any of the foregoing or following, R⁸ isnitrile or —C(O)NH₂. In certain embodiments, —C(O)NH₂ may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl or substituted or unsubstituted cycloalkyl. Incertain embodiments, there is no occurrence of R¹¹.

In certain embodiments of any of the foregoing or following, eachoccurrence of R¹⁰ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted cycloalkyl, such as from —H, or substituted orunsubstituted C₁-C₆ alkyl. In certain embodiments, each occurrence ofR¹⁰ and R¹² is —H. In certain embodiments, there is no occurrence of R⁰and/or R¹².

In certain embodiments of any of the foregoing or following, R⁰ isindependently selected from —H, halogen, hydroxyl, nitro, nitrile,—SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy; R¹ and R² are each independentlyselected from —H, methoxy, fluoro, chloro, bromo, hydroxyl, nitro,nitrile, methyl, or trifluoromethyl;

R³ is selected from methyl, ethyl, propyl, isopropyl, cyclopropyl, orcyclobutyl;

R⁷ and R⁸ are each independently selected from H, —NH₂, —C(O)NH₂,nitrile, methyl, ethyl, isopropyl, cyclopropryl, cyclobutyl,cyclopentyl, cyclohexyl, phenyl, or benzyl; and

A, D, G and K is each independently selected from N or CR¹⁵, whereineach occurrence of R¹⁵ is independently selected from —H, halogen,hydroxyl, nitro, nitrile, substituted or unsubstituted C₁-C₆ alkoxy,substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy.

In certain embodiments of any of the foregoing or following, R⁰ isselected from —H, hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹²;

R¹ and R² are each trifluoromethyl;

R³ is isopropyl;

R⁷ is —NH₂, methyl or phenyl;

R⁸ is nitrile or —C(O)NH₂; and

A, D, G and K is each independently selected from N or CR¹⁵, whereineach occurrence of R¹⁵ is independently selected from —H, halogen,hydroxyl, nitro, nitrile, substituted or unsubstituted C₁-C₆ alkoxy,substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy.

In certain embodiments, the compound is selected from:

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, the R⁰, R¹and R² are not all simultaneously —H. In other embodiments, R⁰ is —H andthe ring to which it is attached, is substituted with a singlesubstituent (other than —H) at one of R¹ or R². In certain embodiments,R⁰ is —H, and R¹ and R² are not —H. In other embodiments, R¹ is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R². In certain embodiments,R¹ is —H, and R⁰ and R² are not —H. In other embodiments, R² is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R¹. In certain embodiments,R² is —H, and R⁰ and R¹ are not —H. In other embodiments, R⁰, R¹ and R²are not —H.

In one aspect, the compounds of the disclosure, or a pharmaceuticallyacceptable salt thereof, are represented by Formula (IX):

wherein:

R⁰, R¹ and R² are each independently selected from —H, halogen,hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹,—OC(O)R¹², —C(O)OR¹², —C(O)R¹⁰R¹², —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted C₁-C₆ haloalkyl, or substituted or unsubstituted C₁-C₆haloalkoxy;

R³ is selected from —H, halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy;

R⁴ is selected from H, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, or substituted or unsubstitutedheteroarylalkyl;

R⁵, R⁶ and R⁷ are each independently selected from —H, —C(O)R¹¹,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl, orsubstituted or unsubstituted heteroarylalkyl, or R⁵ is selected from anyof the foregoing and R⁶ and R⁷ taken together with the nitrogen atom towhich they are attached form a substituted or unsubstituted 3-6 memberedring;

each occurrence of R¹¹ is independently selected from substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl; and

each occurrence of R¹⁰ and R¹² is each independently selected from —H,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocyclyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl.

In some embodiments, the compound is represented by Formula (IXa)(wherein the R groups are as described above for Formula (IX)):

In certain embodiments, the compound is represented by Formula (IXb)(wherein the R groups are as described above for Formula (IX)):

In some embodiments, of any of the foregoing or following, R⁰, R¹ and R²are each independently selected from —H, halogen, hydroxyl, nitro,nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)OR¹², —C(O)R¹¹,—C(O)NR¹⁰R¹², —NR¹⁰R¹², —N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl,substituted or unsubstituted C₁-C₆ haloalkyl, or substituted orunsubstituted C₁-C₆ haloalkoxy.

In some embodiments, of any of the foregoing or following, R⁰ isselected from hydroxyl, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹², —OR¹¹, —C(O)NR¹⁰R¹²,—NR¹⁰R¹², —N(R¹²)C(O)R¹¹, or —NS(O)₂R¹². In other embodiments, R⁰ isselected from —H, halogen, substituted or unsubstituted C₁-C₆ haloalkyl,substituted or unsubstituted C₁-C₆ haloalkoxy, or substituted orunsubstituted C₁-C₆ alkyl. In other embodiments R⁰ is —H.

In some embodiments, of any of the foregoing or following, R¹ and R² areeach independently selected from —H, halogen, hydroxyl, nitro, nitrile,—OR¹¹, substituted or unsubstituted C₁-C₆ alkyl, or substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy. In other embodiments, R⁰ is selected from —H, halogen,substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In some embodiments, of any of the foregoing or following, R¹ and R² areeach independently selected from —H, methoxy, fluoro, chloro, bromo,hydroxyl, nitro, nitrile, methyl, trifluoromethyl, or trifluoromethoxy.In other embodiments, R⁰ is selected from —H, halogen, substituted orunsubstituted C₁-C₆ haloalkyl, substituted or unsubstituted C₁-C₆haloalkoxy, or substituted or unsubstituted C₁-C₆ alkyl. In otherembodiments R⁰ is —H.

In some embodiments, of any of the foregoing or following, R¹ and R² areeach independently selected from H, methoxy, chloro, nitro, nitrile, ortrifluoromethyl. In other embodiments, R⁰ is selected from —H, halogen,substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In some embodiments, of any of the foregoing or following, R¹ and R² areeach trifluoromethyl. In other embodiments, R⁰ is selected from —H,halogen, substituted or unsubstituted C₁-C₆ haloalkyl, substituted orunsubstituted C₁-C₆ haloalkoxy, or substituted or unsubstituted C₁-C₆alkyl. In other embodiments R⁰ is —H.

In certain embodiments of any of the foregoing or following, R³ isselected from —H, halogen, hydroxyl, nitro, nitrile, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocyclyl, substituted or unsubstitutedaryl, or substituted or unsubstituted heteroaryl.

In certain embodiments of any of the foregoing or following, R³ isselected substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl.

In some embodiments of any of the foregoing or following, R³ is selectedfrom methyl, ethyl, propyl, isopropyl, cyclopropyl, or cyclobutyl. Incertain embodiments, any of the foregoing may be optionally substituted.

In some embodiments of any of the foregoing or following, R³ is selectedfrom isopropyl, cyclopropyl, or cyclobutyl. In certain embodiments, anyof the foregoing may be optionally substituted.

In some embodiments of any of the foregoing or following, R³ iscyclobutyl. In certain embodiments, cyclobutyl may be optionallysubstituted.

In certain embodiments of any of the foregoing or following, R⁴ isselected from —H, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, or substituted or unsubstitutedarylalkyl.

In certain embodiments of any of the foregoing or following, R⁴ isselected from —H, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted cycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted arylalkyl.

In some embodiments of any of the foregoing or following, R⁴ is selectedfrom methyl, ethyl, isopropyl, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, phenyl or benzyl. In certain embodiments, any of theforegoing may be optionally substituted.

In some embodiments of any of the foregoing or following, R⁴ is methylor isopropyl. In certain embodiments, any of the foregoing may beoptionally substituted.

In some embodiments, of any of the foregoing or following, R⁴ is methyl.In certain embodiments, methyl may be optionally substituted.

In certain embodiments of any of the foregoing or following, R⁵, R⁶ andR⁷ are each independently selected from —H, —C(O)R¹¹, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedarylalkyl, or R⁵ is selected from any of the foregoing and R⁶ and R⁷taken together with the nitrogen atom to which they are attached form asubstituted or unsubstituted 3-6 membered ring.

In certain embodiments of any of the foregoing or following, R⁵, R⁶ andR⁷ are each independently selected from —H, —C(O)R¹¹, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedarylalkyl.

In some embodiments of any of the foregoing or following, R⁵, R⁶ and R⁷are each independently selected from —H, methyl, ethyl, isopropyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, benzyl, orCOCH₃. In certain embodiments, any of the foregoing, except —H, may beoptionally substituted.

In some embodiments of any of the foregoing or following, R⁵, R⁶ and R⁷are each independently selected from H, methyl, phenyl, or —COCH₃. Incertain embodiments, any of the foregoing, except —H, may be optionallysubstituted.

In some embodiments of any of the foregoing or following, R⁵ and R⁶ areeach independent selected from H, methyl or phenyl. In certainembodiments, any of the foregoing, except —H, may be optionallysubstituted.

In some embodiments of any of the foregoing or following, R⁷ is H.

In some embodiments of any of the foregoing or following, the compoundis selected from:

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, thecompound is selected from the group consisting of

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, thecompound is

or a pharmaceutically acceptable salt thereof. In certain suchembodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, thecompound is,

or a pharmaceutically acceptable salt thereof. In certain suchembodiments, the compound is:

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, thecompound is;

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, thecompound is:

or an enantiomer thereof, or a pharmaceutically acceptable salt of anyone of the foregoing. In certain such embodiment, the compound is:

or a pharmaceutically acceptable salt thereof.

In certain embodiments of any of the foregoing or following, the R⁰, R¹and R² are not all simultaneously —H. In other embodiments, R⁰ is —H andthe ring to which it is attached, is substituted with a singlesubstituent (other than —H) at one of R¹ or R². In certain embodiments,R⁰ is —H, and R¹ and R² are not —H. In other embodiments, R¹ is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R². In certain embodiments,R¹ is —H, and R⁰ and R² are not —H. In other embodiments, R² is —H andthe ring to which it is attached is substituted with a singlesubstituent (other than —H) at one of R⁰ or R¹. In certain embodiments,R² is —H, and R⁰ and R¹ are not —H. In other embodiments, R⁰, R¹ and R²are not —H.

The disclosure also provides variants of Formulae (IV), (V), (VI),(VII), (VIII), and (IX), wherein

contains 1 to 3 heteroatoms (e.g., 1, 2 or 3 heteroatoms) independentlyselected from O or N.

Certain compounds of the disclosure (e.g., Compounds of Formula (IXc),wherein Formula (IXc) is a sub-genus of Formula (IX)) and theircorresponding inhibitory activity values are shown in Table 3 below. Theassay used to evaluate activity is described in the Examples.

TABLE 3 SHMT2 SHMT1 R³ R² R¹ IC₅₀ (nM) IC₅₀ (nM GD-07 —CH₃ —NO₂ —H >5000— HK-1 -cyclobutane —CF₃ —H >5000 — HK-2 -cyclobutane —OMe—H >5000 >5000 HK-3 -cyclobutane —Cl —H 1191 — HK-4 -cyclobutane —CF₃ —H1768 — HK-5 —CH(CH₃)₂ —CF₃ —H 756 — HK-6 —CH(CH₃)₂ —OMe —H 4580 — HK-7—CH(CH₃)₂ —Cl —H 400.5 — HK-8 —CH(CH₃)₂ —CF₃ —H 400.5 — HK-9-cyclobutane —Cl —OMe 3464 — HK-10 -cyclobutane —Cl —Cl 1193 593 HK-11-cyclobutane —Cl —CF₃ 100 — HK-12 -cyclobutane —CF₃ —CF₃ 168 205.7 HK-13—CH(CH₃)₂ —Cl —OMe 500 — HK-14 —CH(CH₃)₂ —Cl —Cl 131 67 HK-15 —CH(CH₃)₂—Cl —CF₃ 35 20 HK-16 —CH(CH₃)₂ —CF₃ —CF₃ 27 21 (racemic mxture) HK-16(PK-1) —CH(CH₃)₂ —CF₃ —CF₃ >5000 >5000 HK-16 (PK-2) —CH(CH₃)₂ —CF₃ —CF₃15 5 HK-X1 —CH(CH₃)₂ —CF₃

66 13 HK-X2 —CH(CH₃)₂ —CH₂OH —C₆H₅ (phenyl) 13 5 HK-X3 —CH(CH₃)₂ —CH₂OH

7.8 16.9 Compound a —CH(CH₃)₂ —CF₃ —Br 42 —

HK-16 (PK2), enriched for an enantiomer of HK-16 (see, for exampleFormula (IXa) and (IXb)), was used in Example 11 and FIG. 4. As shown inExample 11 and FIG. 4, HK-16 (PK2) is not only highly active againstSHMT it also is highly active in terms of its growth inhibitory effecton the tested cancer cell lines.

In certain embodiments, the disclosure provides a pharmaceuticallyacceptable salt of any of the compounds of the disclosure. In certainembodiments, any of the compounds of Formulae (I)-(IX) described herein,or a pharmaceutically acceptable salt thereof, are capable of inhibitingactivity of an SHMT enzyme (e.g., are SHMT inhibitors, such as an SHMT2inhibitor). Any such compounds described based on any of the structuralfeatures described herein may, in certain embodiments, also be describedbased on any of the functional features described herein (e.g., bindingaffinity for SHMT1 and/or SHMT 2, IC50, selectivity, inhibitory effecton serine flux or mitochondrial serine flux, etc.).

In certain embodiments of any of the foregoing or following, a compoundof the disclosure is provided in isolated or substantially purifiedform, such as a substantially purified stereoisomer of a compound of thedisclosure. Without being bound by theory, compounds of the disclosurehave a stereocenter. Thus, in certain embodiments, substantiallypurified stereoisomers are provided and are suitable in any of themethods described herein.

In certain embodiments of any of the foregoing, compounds of thisdisclosure, such as compounds of Formulae (I)-(IX) and others, describedusing any combination of structural and/or functional activity,including any combination of one or more features described above orherein, are provided (and may be provided as an isolated or purifiedform or as a pharmaceutical composition). In certain embodiments, anysuch compounds of the disclosure may be used in any of the methodsdescribed herein, such as to inhibit SHMT activity in vitro or in vivo,or to treat cancer.

In another aspect of any of the foregoing or following, the disclosureprovides a method of inhibiting the activity of a mammalian serinehydroxymethyl transferase (SHMT) enzyme, comprising contacting theenzyme or a cell expressing the enzyme with a compound, or apharmaceutically acceptable salt thereof, or a pharmaceuticalcomposition as described herein. In certain embodiments of any of theforegoing or following, the mammalian SHMT enzyme is a human SHMTenzyme, such as SHMT1, SHMT2, or both. In certain embodiments of any ofthe foregoing or following, the SHMT enzyme is SHMT2.

In certain embodiments of any of the foregoing or following, the methodis an in vitro method comprising contacting the cell with the compound.

In certain embodiments of any of the foregoing, compounds of thisdisclosure, such as those of Formulae (I)-(IX) and others, areinhibitors of SHMT, such as are capable of inhibiting an activity of anSHMT enzyme. In certain embodiments, compounds of Formulae (I)-(IX) areSHMT2 inhibitors and, optionally, are also inhibitors of SHMT1. Incertain embodiments, compounds of Formulae (I)-(IX) are SHMT2inhibitors, but do not inhibit SHMT1. In certain embodiments, compoundsof Formulae (I)-(IX) are dual SHMT inhibitors. In certain embodiments,compounds of Formulae (I)-(IX) inhibit SHMT2 with an IC50 of less than5000 nM and, optionally, inhibit SHMT1 with an IC50 of less than 5000nM. In certain embodiments, compound of Formulae (I)-(IX) inhibit SHMT2with an IC50 of less than 2000 nM, less than 1500 nM, less than 800 nM,less than 500 nM, less than 250 nM, less than 150 nM, or less than 50nM. In certain embodiments, such compounds also inhibit SHMT1 with anIC50 of less than 5000 nM. In certain embodiments, such compoundsinhibit SHMT1 with an IC50 of less than 1000 nM, less than 750 nM, lessthan 500 nM, less than 250 nM, less than 100 nM, or less than 50 nM.

In one aspect, the disclosure further provides pharmaceuticalcompositions comprising one or more compounds of the disclosure togetherwith a pharmaceutically acceptable carrier or excipient. Compounds orpharmaceutical compositions of the disclosure may be used in vitro or invivo. Exemplary compounds of the disclosure, including examples of SHMTinhibitors, are provided herein. SHMT inhibitors may include any suchcompounds, as well as other compounds described structurally and/orfunctionally. The disclosure contemplates compounds have any combinationof any of the foregoing structural and/or functional characteristics.

E. Exemplary Uses

In certain aspects, compounds of the disclosure, such as compounds andcompositions as described herein, can be used for a variety of in vitroor in vivo uses. In other words, in certain embodiments, compounds ofthe disclosure, which may include mammalian SHMT inhibitors, such asSHMT2 inhibitors, such as inhibitors that inhibit both mammalian SHMT2and SHMT1, can be used for a variety of in vitro and/or in vivo uses.Exemplary SHMT inhibitors are described herein. In certain embodiments,the SHMT2 inhibitor is a small organic molecule. In some embodiments,the SHMT2 inhibitor is not an antibody. Compounds of the disclosure(e.g., SHMT inhibitors, such as any of the inhibitors described hereinbased on structure and/or function) are suitable to inhibit SHMTactivity, such as enzyme activity, in vitro or in vivo. In vitro usesinclude for studying SHMT function and/or serine flux and/or folatemetabolism and/or NADPH generation and/or glycine generation in, forexample, healthy cells, cancerous cells and/or in hypoxic cells.Similarly, folate metabolism, NADPH generation, and/or SHMT activity canbe assessed in mutant cell lines, such as cells in which the activity ofMTHFD1, MTHFD1L, MTHFD2, MTHFD2L, SHMT1, SHMT2, MTHFR, ALDH1L1, ALDHL2,FH, KEAP1 is or has been inhibited, disregulated or knocked out, orcells having certain hyperactivating mutations in any of the foregoingor in NRF2. Evaluation of compounds in such mutant or knock out celllines, or in cell lines harboring mutations affecting mitochondrialmetabolism or a mitochondrial folate pathway is also useful foridentifying cell types and cancer types in which compounds of thedisclosure would be particularly useful, have increasedanti-proliferative activity and/or improved activity at a lower dose.This can be seen in the examples.

In vivo uses include for studying SHMT activity and/or folate metabolismand/or serine flux and/or NADPH generation in animal models of disease,such as in animal cancer models, such as mouse xenografts. In certainembodiments, compounds of the disclosure are useful for evaluating theimpact of hypoxic conditions to growth, survival and migration of cells,such as the tolerance of cells to hypoxic conditions. In certainembodiments, the cells or animal model comprise a mutation in Myc.

Suitable in vivo uses include treating cancer or an autoimmunecondition. For example, suitable in vivo uses include treating acondition associated with SHMT activity and/or associated withalterations in mitochondrial metabolism, such as mitochondrial folatemetabolism. For example, compounds of the disclosure, such as SHMT2inhibitors, or such as any of the compounds described herein based onstructure and/or function, are suitable for use in conditions in whichincreased mitochondrial activity is necessary or useful for diseaseprogression, and/or SHMT expression or activity (e.g., SHMT2) iselevated in the disease state versus the healthy state and/or in whichthere is an alteration in mitochondrial metabolism, such asmitochondrial folate metabolism (e.g., via mutations or alterations in,for example, cancer cells).

Exemplary genes or proteins that may be disregulated in, for example,cancer cells, are described above and include, for example, MTHFD1,MTHFD1L, MTHFD2, MTHFD2L, SHMT1, SHMT2, MTHFR, ALDH1L1, ALDHL2, SLC25A32(also known as the mitochondrial folate transporter MFT), FH, KEAP1,and/or NRF2. Accordingly, the disclosure provides methods of treatingcancer in a mammalian subject in need thereof, such as by inhibitingSHMT in a mammalian subject in need thereof, such as a subject with acondition associated with SHMT activity and/or associated withalterations in mitochondrial metabolism, such as mitochondrial folatemetabolism.

In certain embodiments, the disclosure provides compounds, compositions,and methods of treating malignancies (such as malignancies of B-cellorigin, e.g., cancer of B-cell origin) in which the SLC38A2 gene isdisregulated (e.g., down-regulated). In certain embodiments, thedisclosure provides compounds, compositions, and methods of treatinglymphoma (e.g., B-cell lymphoma) in which the SLC38A2 gene isdisregulated (e.g., down-regulated). Moreover, and as shown in the FIG.13, we observed that decreased expression of SLC38A2, as assessed bymRNA expression, is correlated with sensitivity to a combination of anSHMT inhibitor+formate.

Accordingly, the disclosure provides methods of treating lymphomas in asubject in need thereof, such as B-cell lymphomas, wherein the lymphomais characterized by decreased expression of SLC38A2 (e.g., relative tohealthy control cells, such as control cells of the same or an adjacentcell type from the same person or against a standard). In certainembodiments, the patient in need thereof is a patient having a lymphomathat can be characterized by decreased expression of SLC38A2, such as aB-cell lymphoma, such as diffuse large B-cell lymphoma having suchdecreased expression. In certain embodiments, the expression of SLC38A2is mRNA expression. In certain embodiments, the expression is proteinexpression. Without being bound by theory, such cancers having a loss ofor decreased expression of SLC38A2 represent a subset of B-celllymphomas and identify a distinct patient population particular suitablefor and sensitive to treatment with a combination of an (i) SHMTinhibitor (SHMT 1, SHMT2, or dual inhibitor) and (ii) formate, folinicacid, or a derivative or related compound thereof, as described herein.

In certain embodiments, the method comprises administering atherapeutically effective amount of a compound of the disclosure. Incertain embodiments, administration of the compound improves one or moresymptoms of the disease or condition. In the case of cancer, suchimprovement in symptoms may include, for example, decrease in tumorsize, decrease in disease progression, increased time to progression,increased overall patient survival, decrease in metastasis or decreasein time to metastasis, and the like. In certain embodiments, improvementin these symptoms is evaluated versus a control (e.g., an untreatedpatient or a patient receiving the standard of care).

Similarly, as demonstrated in the examples, compounds of the disclosure,such as SHMT inhibitors are useful to treat conditions in which there isa mutation or dysfunction in mitochondrial metabolism, such as amitochondrial folate pathway. These may include activating mutations orupregulating of enzymes in the pathway. However, this may also includeloss of function mutations that lead to dysregulation. Without wishingto be bound by theory, such dysregulation may sensitize cells to SHMTinhibitors, particularly inhibitors of the present disclosure capable ofinhibiting both SHMT2 and SHMT1. Such conditions are not the onlyconditions in which compounds of the disclosure are effective (as shownherein). However, they may represent a category of conditions that areparticularly sensitive to this approach.

In certain embodiments, the disclosure provides a method for treatingcancer. In certain embodiments, the cancer is associated with SHMTactivity. In certain embodiments, the method comprises administering aneffective amount of a compound or composition of the disclosure, such asan SHMT2 inhibitor, such as any of the compounds described herein basedon structure and/or function. SHMT inhibitors may be administered as amonotherapy or in combination with one or more additional agents ortherapeutic modalities as part of a therapeutic regimen. In certainembodiments, administration of the compound (alone or as part of thetherapeutic regimen) improves one or more symptoms of the disease orcondition. In the case of cancer, such improvement in symptoms mayinclude, for example, decrease in tumor size, decrease in pain, decreasein disease progression, increased time to progression, increased overallpatient survival, decrease in metastasis or decrease in time tometastasis, and the like. In certain embodiments, improvement in thesesymptoms is evaluated versus a control (e.g., an untreated patient or apatient receiving the standard of care).

In certain embodiments, this disclosure provides a method for treatinglymphoma, such as T-cell lymphoma, B-cell lymphoma, or NK-cell lymphoma,comprising administering to a mammalian subject in need thereof aneffective amount of an SHMT inhibitor. In certain such embodiments, thelymphoma is a B-cell lymphoma. In certain embodiments, the B-celllymphoma is diffuse large B-cell lymphoma or Burkitt lymphoma. Incertain embodiments, the B-cell lymphoma is characterized as having thefollowing in vitro activity: growth sensitivity to an SHMT inhibitor invitro that is not rescuable by formate but is rescuable by a combinationof formate and glycine, wherein growth sensitivity is assessed by cellcount in vitro. In some embodiments, the glycine rescue is sufficient torestore glycine levels in the normal body (e.g., a healthy cell ortissue) but not in the tumor or lymphoma.

In certain embodiments, the disclosure provides a method for treating anautoimmune disorder. In certain embodiments, the autoimmune disorder isassociated with SHMT activity. In certain embodiments, the methodcomprises administering an effective amount of a compound or compositionof the disclosure, such as an SHMT2 inhibitor, or any of the inhibitorscompounds herein based on structure and/or function. SHMT inhibitors maybe administered as a monotherapy or in combination with one or moreadditional agents or therapeutic modalities as part of a therapeuticregimen. In certain embodiments, administration of the compound (aloneor as part of the therapeutic regimen) improves one or more symptoms ofthe disease or condition. In the case of an autoimmune disorder, suchimprovement in symptoms may include, for example, decrease ininflammatory markers, decrease in inflammation, decrease in pain,increased mobility and/or range of motion, and improvements in patientreports on quality of life measures. The particular symptoms improvedwill vary based on the autoimmune condition. In certain embodiments,improvement in these symptoms is evaluated versus a control (e.g., anuntreated patient or a patient receiving the standard of care).

In certain aspects, the disclosure contemplates that any of thecompounds of the disclosure (and pharmaceutical compositions) may beused in any of the in vitro or in vivo methods provided herein. Incertain embodiments, compounds of the disclosure are SHMT inhibitors andare suitable for inhibiting SHMT activity, such as SHMT2 and,optionally, SHMT1 activity. In certain embodiments, such compounds aresuitable for modulating SHMT activity in vitro, such as to manipulateserine flux and/or folate metabolism. Such in vitro methods may beuseful for identifying other components of folate metabolic pathways. Inother embodiments, such compounds are suitable for modulating SHMTactivity in vivo, such to treat a patient suffering from a SHMT-relatedcondition or a condition that can be ameliorated by inhibiting SHMTactivity and/or a condition associated with alterations in mitochondrialmetabolism, such as mitochondrial folate metabolism, such as cancer andautoimmune disorders.

Moreover, any of the compounds of the disclosure may be formulated as apharmaceutical composition comprising a compound and one or moreacceptable carriers and/or excipients. Compositions, such aspharmaceutical compositions, may be used in any of the in vitro or invivo methods described herein, such has to treat any one or more of thediseases or conditions described herein.

Accordingly, the disclosure contemplates methods of treating (decreasingthe frequency or severity of or otherwise alleviating one or moresymptoms of the condition) a subject in need thereof (e.g., a subjecthaving any of the conditions described herein, including any of theautoimmune conditions described herein or any of the forms of cancerdescribed herein) by administering a compound of the disclosure (e.g.,SHMT inhibitors, such as any of the inhibitors described herein based onstructure and/or function), such as an effective amount of a compound ofthe disclosure.

Cancers and Proliferative Disorders

In certain embodiments, compounds and compositions of the disclosure areuseful to treat cancer, such as to reduce cancer cell growth, survivaland/or metastasis. Such cancers include, for example, solid tumors andhematological malignancies (both adult and pediatric). Exemplary cancersinclude, but are not limited to, leukemia (including, but not limitedto, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia(AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia(CML), acute monocytic leukemia (AMoL)), lymphoma (non-Hodgkin'slymphoma or Hodgkin's lymphoma), lung cancer (including non-small lungcancer), mesothelioma, breast cancer (including other solid tumors ofthe breast), liver cancer (including other solid tumors of the liver),colon or colorectal cancer (including other solid tumors of the colonand/or rectum), stomach cancer (including other solid tumors of thestomach), prostate cancer (including other solid tumors of theprostate), pancreatic cancer (including other solid tumors of thepancreas), ovarian cancer (including other solid tumors of the ovary),solid tumors of the uterus or female genital tract, bladder cancer(including other solid tumors of the bladder), head and neck cancers,glioblastoma and other brain tumors, and trophoblastic neoplasms. Incertain embodiments, the cancer is a lymphoma, preferably a B-celllymphoma. In certain embodiments, the B-cell lymphoma is a diffuse largeB-cell lymphoma or a Burkitt lymphoma.

Although the high metabolic needs of cancer cells make all cancers goodcandidates for treatment with SHMT inhibitors, certain cancers may beparticularly susceptible to treatment or may be sensitized to treatmentdue to their underlying mitochondrial activity or mutational status. Byway of non-limiting example, in certain embodiments, the cancercomprises cells having a mutation affecting mitochondrial metabolism orthe mitochondrial folate pathway. One class of cancers has highexpression levels of SHMT2 or upregulation in a component of themitochondrial folate pathway. However, other mutations may impairmitochondrial metabolism or the mitochondrial folate pathway, and thus,sensitize cancers to SHMT inhibition. Examples of such sensitization areprovided herein. Examples of such sensitization are provided herein.Exemplary genes or proteins that may be disregulated in, for example,cancer cells, are described above and include, for example, MTHFD1,MTHFD1L, MTHFD2, MTHFD2L, SHMT1, SHMT2, MTHFR, ALDH1L1, ALDHL2, SLC25A32(also known as the mitochondrial folate transporter MFT), FH, KEAP1,and/or NRF2.

Other classes of cancers that may be particularly susceptible totreatment with an SHMT inhibitor are cancers comprising mutations thatinactivate KEAP1 (either by, for example, somatic mutation or epigeneticsilencing). Such mutations may result in aberrant NRF2 activity andnuclear function. Additionally or alternatively, suitable cancers mayadditionally have mutations in NRF2 itself with or without KEAP1mutation. Cancers may additionally have alterations in mitochondrialmetabolism including mutations to fumarate hydratase (FH) that lead toactivation of NRF2. Loss of FH activity may potentiate cells to SHMTinhibitors by additional mechanisms as well. Without being bound bytheory, cancers may have aberrant activation of NRF2 via othermechanisms. Aberrant NRF2 activation leads to altered transcription ofmitochondrial and one carbon metabolism genes through the activity ofATF4. Activation of the 1c pathway and mitochondrial folate metabolismby NRF2 may also occur via ATF4 independent mechanisms.

Exemplary cancers with identified mutations in the KEAP1-NRF2-ATF4pathway include non-small cell lung cancer, squamous cell lungcarcinoma, prostate cancer, head and neck cancer (KEAP1 mutations, NRF2mutations), hereditary papillary renal carcinoma, Hereditaryleiomyomatosis and renal cell cancer (FH mutations). Furthermore, asdescribed herein, there are identified cancers with elevated SHMT2levels and/or mutations in other folate pathway components.

Without being bound by theory, the present disclosure provides methodsfor treating cancer and autoimmune conditions, such as cancers andconditions associated with SHMT activity. In some embodiments,particularly susceptible cancers or autoimmune conditions are those inwhich SHMT2 or another mitochondrial folate pathway enzyme or anothergene is upregulated or activated. In other embodiments, particularlysusceptible cancers or autoimmune conditions are those in which SHMT2 oranother mitochondrial folate pathway component are knocked out ordownregulated, thereby increasing the susceptibility of cells totreatment.

In certain embodiments, a compound of the disclosure is administered asa monotherapy. In other embodiments, compounds of the disclosure areused in combination with one or more other agents and/or therapeuticmodalities (e.g., dietary regimen). When more than one agent isadministered, the agents may be administered at the same or varyingtimes, and by the same or differing routes of administration.

In certain embodiments, compounds of the disclosure are used incombination with the then current standard of care of the particularcondition as part of a therapeutic regimen. In other embodiments, thetherapeutic regimen includes one or more antifolates (e.g., other than acompound of the disclosure; an antifolate that is not selective forSHMT1 and/or SHMT2), such as traditional antifolates, such asmethotrexate or pemetrexed or another compound that is an inhibitor ofDHFR and/or TS. In other embodiments, the therapeutic regimen includesan additional anti-cancer agent, such as 5FU or other chemotherapeuticor radiotherapy regimen. In certain embodiments, the therapeutic regimenincludes rescue therapy, such as leucovorin, formate or pharmaceuticalsalts, esters or derivatives of formate. In certain embodiments, rescuetherapy refers to administration of folinic acid (leucovorin), formateand/or a pharmaceutical salt of formate. In certain embodiments,administration of rescue therapy provide rescue of SHMT activity innon-cancerous tissues, reducing systemic toxicity and increasingtherapeutic index. In certain embodiments, such as in the context of a Bcell neoplasia, the inhibition activity of an SHMT inhibitor may beenhanced when administered in combination with rescue therapy, such asformate, glycine, folinic acid (leucovorin) or a pharmaceuticallyacceptable salt, ester, or derivative thereof.

Without being bound by theory, use of rescue therapy to reduce toxicityand/or side effects may be used when SHMT inhibitors are used alone, aswell as when they are used as part of a therapeutic regimen with one ormore additional agents or therapeutic modalities. Rescue therapy isroutinely used currently in patients receiving, for example,methotrexate. Accordingly, its use can be readily adapted to thiscontext. For example, the dose of rescue therapy can be tittered todecrease toxicity without abrogating therapeutic efficacy. Similarly,rescue therapy may be administered at the same time, or followingadministration of an SHMT inhibitor to manage any toxicity (if any)while maintaining an acceptable therapeutic profile. In otherembodiments, use of rescue therapy is unnecessary because the safety andtoxicity profile is acceptable.

In certain embodiments, co-administration results in an additive orsynergistic effect versus administration of at least one of thecompounds alone. In certain embodiments, co-administration permitsadministration of a lower dose of the non-SHMT inhibitor compound (e.g.,effectiveness is reached at a lower dose of, for example, methotrexate)or of a higher dose of the SHMT inhibitor or other agent by, forexample, reducing side effects. In certain embodiments, the combinationtherapy improves the therapeutic window of one or both compounds orreduces side effects associated with one or both compounds.

In other embodiments, a compound of the disclosure is administered witha glycine containing composition. In other embodiments, the therapeuticregimen includes a dietary regimen, such as a regimen in which dietarylevels of methionine, serine, and/or choline are reduced and/or dietarylevels of glycine are increased.

In certain embodiments, the cancer is pancreatic cancer or colon cancer.In certain embodiments, the cancer is a cancer comprising (e.g., inwhich one or more cells of the tumor or cancer have) a mutation in themitochondrial folate pathway. For example, in certain embodiments, thecompounds of the disclosure, such as SHMT inhibitors are used to treat acancer comprising a mutation in the mitochondrial folate pathway, suchas mutation in mitochondrial serine hydroxymethyl transferase (SHMT2),mitochondrial methylene tetrahydrofolate dehydrogenase (MTHFD2), MTHFD1Lor FH.

The disclosure contemplates combinations of any of the foregoingembodiments and aspects. In other words, the disclosure contemplatesthat any of the compounds of the disclosure, including any inhibitor ofmammalian SHMT2 (and, in certain embodiments also SHMT1), may be used inany of the in vitro or in vivo methods, including methods of treatment.Moreover, such compounds and pharmaceutical compositions may be used asa monotherapy or as part of a therapeutic regimen with one or more otheragents or treatment modalities, including but not limited tochemotherapy, anti-folate agent, rescue therapy, glycine, radiationtherapy, nutritional therapy, and/or the standard of care for theparticular cancer. Exemplary categories of compounds for use in treatingcancer or autoimmune condition are, for example, Compounds of Formulae(I)-(IX), or any of the compounds described based on a combination ofstructural and/or functional characteristics herein.

Autoimmune Conditions

In other embodiments, the disclosure provides methods of treating anautoimmune condition by administering a compound of the disclosure. Suchdiseases or disorders include, but are not limited to, rheumatoidarthritis, multiple sclerosis, systemic lupus erythematosus, myastheniagravis, inflammatory bowel disease (e.g., Crohn's disease or ulcerativecolitis), polymyositis, dermatomyositis, inflammatory myositis,ankylosing spondolytis, and ulcerative colitis. Similar to as describedabove for cancer, autoimmune disorders, as well as other conditions, maybe treated based on administering a compound of the disclosure as amonotherapy or as a combination therapy, as described above.

As noted above, in certain embodiments, compounds of the disclosure maybe particularly suitable for use in subjects having one or moremutations that impact mitochondrial metabolism, as described above. Suchas contemplated here. Additionally or alternatively, in autoimmuneconditions the mitochondrial folate pathway may be activated, and thus,compounds of the disclosure offer a mechanism for regulating thisinappropriate activation or dysregulation of mitochondrial metabolism.

The disclosure contemplates combinations of any of the foregoingembodiments and aspects. In other words, the disclosure contemplatesthat any of the compounds of the disclosure, including any inhibitor ofmammalian SHMT2 (and, in certain embodiments also SHMT1), may be used inany of the in vitro or in vivo methods, including methods of treatment.Moreover, such compounds and pharmaceutical compositions may be used asa monotherapy or as part of a therapeutic regimen with one or more otheragents or treatment modalities, including but not limited tochemotherapy, anti-folate agent, rescue therapy, glycine, radiationtherapy, nutritional therapy, and/or the standard of care for theparticular cancer. Exemplary categories of compounds for use in treatingcancer or autoimmune condition are, for example, compounds of Formulae(I)-(IX), or any of the compounds described based on a combination ofstructural and/or functional characteristics herein.

F. Compositions and Modes of Administration

In some embodiments of this disclosure, a compound of the presentdisclosure is formulated with one or more pharmaceutically acceptablecarriers, excipients and/or solvents. The disclosure provides suchcompositions and pharmaceutical compositions. Any of the compounds ofthe disclosure may be provided in isolated or purified form and/or as apharmaceutical composition. The compound may be formulated foradministration in any convenient way for use in human medicine. Anycompound of the disclosure or salt or enantiomer thereof can be providedas a composition, such as a pharmaceutical composition, such as acomposition having any of the features described herein. Any suchcompound of the disclosure or composition of the disclosure may be usedin any of the in vitro or in vivo methods described herein.

Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The characteristics of the carrier will depend on the route ofadministration. Each of the methods or uses of the present disclosure,as described herein, comprises administering to a mammal in need of suchtreatment or use an effective amount, such as a pharmaceutically ortherapeutically effective amount, of a compound of the disclosure, or apharmaceutically acceptable salt thereof. Compounds may be administeredalone or in combination with other agents.

Compounds or pharmaceutical compositions of the disclosure may beadministered to cells in vitro, such as by addition to culture media.Additionally or alternatively, compounds or pharmaceutical compositionsmay be administered to route of administration, such as oral,parenteral, intravenous, intra-arterial, cutaneous, subcutaneous,intramuscular, topical, intracranial, intraorbital, ophthalmic,intravitreal, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, central nervoussystem (CNS) administration, or administration by suppository. In someembodiments, the therapeutic methods of the disclosure includeadministering the composition of a compound topically, systemically, orlocally. For example, therapeutic compositions of compounds of thedisclosure may be formulated for administration by, for example,injection (e.g., intravenously, subcutaneously, or intramuscularly),inhalation or insufflation (either through the mouth or the nose) ororal, buccal, sublingual, transdermal, nasal, or parenteraladministration. The compositions of compounds described herein may beformulated as part of an implant or device, or formulated for slow orextended release. When administered parenterally, e.g., by intravenous,cutaneous or subcutaneous injection, the therapeutic composition ofcompounds for use in this disclosure is preferably in a pyrogen-free,physiologically acceptable form.

A preferred pharmaceutical composition for intravenous, cutaneous, orsubcutaneous injection should contain, in addition to thetoxicity-reducing compounds, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the compound of thepresent disclosure may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.Techniques and formulations generally may be found in Remington'sPharmaceutical Sciences, Meade Publishing Co., Easton, Pa.

A composition comprising a compound of the present disclosure may alsocontain adjuvants, such as preservatives, wetting agents, emulsifyingagents and dispersing agents. Prevention of the action of microorganismsmay be ensured by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption, suchas aluminum monostearate and gelatin.

When an effective amount of a compound(s) of the present disclosure isadministered orally, compound(s) of the present disclosure may be in theform of a tablet, capsule, powder, solution or elixir. When administeredin tablet form, the pharmaceutical composition may additionally containa solid carrier such as a gelatin or an adjuvant. The tablet, capsule,and powder may contain from about 5 to 95% of a compound of the presentdisclosure, and preferably from about 10% to 90% of a compound of thepresent disclosure. When administered in liquid form, a liquid carriersuch as water, petroleum, oils of animal or plant origin such as peanutoil, mineral oils, phospholipids, tweens, triglycerides, includingmedium chain triglycerides, soybean oil, or sesame oil, or syntheticoils may be added. The liquid form of the pharmaceutical composition mayfurther contain physiological saline solution, dextrose or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol. When administered in liquid form, thepharmaceutical composition typically contains from about 0.5 to 90% byweight of a compound of the present disclosure, and preferably fromabout 1 to 50% by weight of a compound of the present disclosure.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more compositionscomprising the compound of the present disclosure may be mixed with oneor more pharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the compound of the present disclosure, theliquid dosage forms may contain inert diluents commonly used in the art,such as water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol (ethanol), isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, coloring, perfuming, and preservativeagents.

The pharmaceutical compositions may be in the form of a liposome ormicelles in which the toxicity-reducing compounds are combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728;4,837,028; and 4,737,323, all of which are incorporated herein byreference.

Suspensions, in addition to the active compounds may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The amount of compound(s) of the present disclosure in thepharmaceutical composition will depend upon the nature and severity ofthe condition being treated, on the amount of the compound of thepresent disclosure used, and on the nature of prior treatments thepatient has undergone. Ultimately, the practitioner will decide theamount of the compound of the present disclosure with which to treateach individual patient. Representative doses of the present disclosureinclude, but are not limited to, about 0.001 mg to about 5000 mg, about0.001 mg to about 2500 mg, about 0.001 mg to about 1000 mg, 0.001 mg toabout 500 mg, 0.001 mg to about 250 mg, about 0.001 mg to 100 mg, about0.001 mg to about 50 mg and about 0.001 mg to about 25 mg. Multipledoses may be administered during one day, especially when relativelylarge amounts are deemed to be needed. It is contemplated that thevarious pharmaceutical compositions used to practice the methods of thepresent disclosure should contain about 0.1 μg to about 100 mg(preferably about 0.1 mg to about 50 mg, more preferably about 1 mg toabout 2 mg) of compound per kg body weight.

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, as2,3,4 or more sub-doses per day. The sub-dose itself may be furtherdivided, e.g., into a number of discrete loosely spaced administrations.The daily dose can be divided, especially when relatively large amountsare administered as deemed appropriate, into several, for example 2, 3or 4 part administrations. If appropriate, depending on individualbehavior, it may be necessary to deviate upward or downward from thedaily dose indicated.

This disclosure will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the disclosure as described more fully in the embodiments whichfollow thereafter.

EXEMPLIFICATION

The subject matter of this disclosure now being generally described, itwill be more readily understood by reference to the following exampleswhich are included merely for purposes of illustration of certainaspects and embodiments of the present disclosure, and are not intendedto limit the subject matter of this disclosure.

Example 1: General Synthetic Scheme

The compounds of this disclosure may be prepared in general by methodsknown to those skilled in the art. Schemes 1-3 below illustrate generalsynthetic routes to the compounds of the present disclosure according tocertain embodiments. Other equivalent schemes, which will be readilyapparent to the ordinary skilled organic chemist, may alternatively beused to synthesize various portions of the molecules as illustrated bythe general scheme below.

Compounds of Formulae (I), (Ia or Ib); (II), (IIa or IIb); (III) (IIIaor IIIb); or (II′) (IIa′ or IIb′) can be prepared according to thefollowing Schemes 1-3.

Example 2: Preparation of6-amino-4-(3-bromo-5-(trifluoromethyl)phenyl)-4-isopropyl-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(Compound a) Via the Following Scheme 4

Scheme 4. Reagents and conditions: (1) carbonyldiimidazole,methoxymethylamine, dichloromethane, room temperature, 18 h; (2)i-PrMgCl, Et₂O, room temperature, overnight; (3) CHCl₃, malodinitrile,Ti(Cl)₄, pyridine, reflux, 48 h; (4) EtOH/dioxane (1:1), piperidine,100° C./microwave, 12 h.

A solution of compound a (500 mg, 1.4 mmol) and3-hydroxy-5-methylpyrazole (286.5 mg, 2.9 mmol) in ethanol (5 ml)dioxane (5 ml) and piperidine (12.4 mg, 0.146 mmol) is heated for 12 hat 100° C. in the microwave. After cooling water is added and extractedwith ethylacetate. The organic phase is dried with sodium sulfate andthe solvents evaporated. The residue was purified by pre-chromatographedon silica with PE/EA=1:1 to yield 0.66 g of (a) (24% yield) as whitesolid which was used for the next step.

Example 3: Preparation of Compound 61, 2, 4, 8, 11, 69, 70, 71, 72, and73

To a solution of Compound a (300 mg, 679.9 umol) in trietheyl amine (9mL) and DMF (3 mL) at 50° C. was added Ph(PPh₃)₂Cl₂ (47.7 mg, 67.9 umol)and CuI (25.9 mg, 135.9 umol). The reaction mixture was slowly warmed to100° C. before the addition of pent-4-yn-1-ol (74.3 mg, 883.8 umol). Thereaction mixture was allowed to stir at 100° C. for 12 hours until LCMSindicated formation of the product. The solvent as removed in vacuo togive the crude residue. The crude residue was purified by pre-thin layerchromatography on silica (DCM:MeOH=20:1) and pre-HPLC (column: Waters,XBridge 150×25 5μ; mobile phase: solvent A: NH₄HCO₃ aqueous solution (10mM); solvent B: acetonitrile; gradient B %: 30-45%) to give the product6-amino-4-(3-(5-hydroxypent-1-yn-1-yl)-5-(trifluoromethyl)phenyl)-4-isopropyl-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrileCompound 61 (87.5 mg, 28.9%) as white solid when solidified. ¹H NMR(CDCl₃, 400 MHz), 59.19 (s, 1H), 7.58 (s, 1H), 7.54 (s, 1H), 7.50 (s,1H), 4.75 (s, 2H), 3.82 (t, J=6.4 Hz, 2H), 2.82-2.78 (m, 1H), 2.55 (t,J=14.0 Hz, 2H), 1.91-1.86 (m, 2H), 1.83 (s, 3H), 1.26 (s, 1H), 1.02 (d,J=6.8 Hz, 2H), 3.82 (d, J=6.4 Hz, 2H). LC-MS (m/z): 445 [M+H]⁺.

Compounds 2, 4, 8, 71, and 73 were prepared in a manner analogous tothat shown above for Compound 61.

Compound 2 was prepared with Compound a reacting with 3-butyn-1-ol.

Compound 4 was prepared with Compound a reacting with ethyl ester of1-pentyn-5-carboxylic acid, followed by hydrolysis using LiOH/THF.

Compound 8 was prepared with Compound a reacting with mono Boc-protected5-pentyn-1-amine, followed by TFA deprotection of the Boc group to yieldthe HCl salt of Compound 8 as a white solid salt after purification.

Compound 71 was prepared with6-amino-4-(3-bromo-5-cyanophenyl)-4-isopropyl-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile(a derivative of Compound a) as the starting material.

Compound 73 was prepared with Compound a reacting with 1-pentyn-5-ethylester. Compound 73 was isolated as a white solid after purification.

Compounds 11, 69, 70, and 72 were prepared with Compound 4 as thestarting material and their preparations are described as follows:

Compound 11 was prepared with Compound 4 reacting with ammoniumhydroxide under the amide coupling reaction conditions with HATU.Compound 11 was purified in the form of a HCl salt as a while solid.

Compound 69 was prepared with Compound 4 reacting with t-butyl-esterprotected (S)-glutamic acid under amid coupling reaction conditions withHATU, followed by acid deprotection of the t-butyl groups using TFA.Compound 69 was purified in the form of a HCl salt as a white solid.

Compound 70 was prepared following similar protocol as Compound 69 usingthe t-butyl-ester protected (R)-glutamic acid. Compound 70 was purifiedin the form of a HCl salt as a white solid.

Compound 72 was prepared with Compound 4 reacting with t-butyl-esterprotected dimethyl amino acid followed by acid deprotection of thet-butyl group TFA. Compound 72 was purified in the form of a HCl salt asa white solid.

Characterizations of exemplary compounds of this disclosure are includedin Table 4 below.

TABLE 4 Com- LC-MS pound (m/z) # [M + H]⁺ NMR Characterization 61 445.1¹H NMR (d⁶-DMSO, 300 MHz), δ 12.24 (S, 1H), 7.61(S, 1H), 7.56(S, 1H),7.52(S, 1H), 7.01(S, 1H), 4.54(t, J = 5.4 Hz, 1H), 3.43 (m, 2H), 2.75(m, 1H), 2.52-2.46(m, 2H), 1.74-1.67 (m, 5H), 0.89 (d, J = 6 Hz, 3H),0.76 (d, J = 6 Hz, 3H) 2 472.1 ¹H NMR (CD₃OD, 300 MHz), δ 7.68 (s, 1H),7.59- 7.58 (m, 2H), 3.75 (t, J = 6 Hz, 2H), 2.84(m, 1H), 2.68(t, J = 6Hz, 2H), 1.80 (s, 3H), 1.02 (d, J = 6 Hz, 3H), 0.89 (d, J = 6 Hz, 3H) 4459.1 ¹H NMR (d⁶-DMSO, 300 MHz), δ 12.25 (S, 1H), 7.59(S, 1H), 7.55(S,1H), 7.53(S, 1H), 7.01(S, 1H), 2.77 (m, 1H), 2.65-2.60(m, 2H),2.51-2.45(m, 2H), 1.70 (s, 3H), 0.88 (d, J = 6 Hz, 3H), 0.76 (d, J = 6Hz, 3H) 8 444.2 ¹H NMR (d⁶-DMSO, 300 MHz), δ 12.27 (S, 1H), 7.80 (br s,2H), 7.65(S, 1H), 7.57(m, 2H), 7.03(S, 2H), 2.94-2.90 (m, 2H), 2.78-2.74(m, 1H), 2.60- 2.50(m, 2H), 1.86-1.81(m, 2H), 1.70 (s, 3H), 0.89 (d, J =6 Hz, 3H), 0.77 (d, J = 6 Hz, 3H) 11 458.1 ¹H NMR (d⁶-DMSO, 300 MHz), δ12.24 (S, 1H), 7.59(s, 1H), 7.55(s, 1H), 7.53(s, 1H), 7.39(s, 1H),7.06(br s, 2H), 6.96(br s, 1H), 2.80-2.73 (m, 1H), 2.63(t, J = 7.2 Hz,2H), 2.39(t, J = 7.2 Hz, 2H), 1.70 (s, 3H), 0.89 (d, J = 6 Hz, 3H), 0.76(d, J = 6 Hz, 3H) 69 588.2 ¹H NMR (d⁶-DMSO, 300 MHz), δ12.25 (S, 1H),8.24(d, J = 7.5 Hz, 1H), 7.61(s, 1H), 7.58(s, 1H), 7.51(s, 1H), 7.01(brs, 2H), 4.25-4.21(br s, 1H), 2.79-2.75 (m, 1H), 2.67-2.63(m, 2H),2.51(m, 2H), 2.33-2.25 (m, 2H), 1.99-1.96(m, 1H), 1.79-1.70(m, 1H), 1.70(s, 3H), 0.89 (d, J = 6 Hz, 3H), 0.76 (d, J = 6 Hz, 3H) 70 588.2 ¹H NMR(CD₃OD, 300 MHz), δ 7.70(s, 1H), 7.58(s, 1H), 4.52-4.47(m, 1H),2.88-2.81(m, 1H), 2.79- 2.71(m, 2H), 2.60-2.55(m, 2H), 2.44-2.39 (m,2H), 2.27-2.18(m, 1H), 2.00-1.90(m, 1H), 1.80 (s, 3H), 1.03 (d, J = 6Hz, 3H), 0.90 (d, J = 6 Hz, 3H) 71 402.3 ¹H NMR (d⁶-DMSO, 300 MHz), δ12.23 (S, 1H), 7.76(br S, 2H), 7.53(S, 1H), 7.09(S, 1H), 3.50 (t, J = 6Hz, 2H), 2.75 (m, 1H), 2.52-2.45(m, 2H), 1.71(s, 3H), 1.70-1.67 (m, 2H),0.89 (d, J = 6 Hz, 3H), 0.76 (d, J = 6 Hz, 3H) 72 544.2 ¹H NMR (CD₃OD,300 MHz), δ 7.65 (s, 1H), 7.59- 7.57 (m, 2H), 2.87-2.85(m, 1H), 2.81(t,J = 6 Hz, 2H), 2.70(t, J = 6 Hz, 2H), 1.78 (s, 3H), 1.46 (s, 6H), 1.02(d, J = 6 Hz, 3H), 0.89 (d, J = 9 Hz, 3H) 73 473.1 ¹H NMR (d⁶-DMSO, 300MHz), δ 12.24 (S, 1H), 7.60(br S, 1H), 7.55(br S, 2H), 7.01(S, 2H), 3.63(s, 3), 2.82-2.65 (m, 5H), 1.70 (s, 3H), 0.89 (d, J = 6 Hz, 3H), 0.76(d, J = 6 Hz, 3H)

Example 4: Preparation of Compound 62:6-amino-4-(3-(5-bromopent-1-yn-1-yl)-5-(trifluoromethyl)phenyl)-4-isopropyl-3-methyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile

In a 10 mL round bottom flask, PPh₃ (70.8 mg, 270 μmol) was dissolved inκ mL of anhydrous dichloromethane (DCM) under N₂ protection. Aftercooling the solution to 0° C., Br₂ (43.1 mg, 270 μmol) was added slowlyand the reaction mixture was stirred for 30 min at 0° C. before slowlyadding Compound 61 (100 mg, 225 μmol). The reaction mixture was allowedto warm to 25° C. and stirred for 5 hours until LC-MS showed theformation of the product. The reaction mixture was then concentrated invacuo and compound 62 (30 mg, crude) was obtained as light yellow solid(30 mg) and used directly in the next reaction. LC-MS (m/z): 508 [M+H]⁺.

Example 5: Labeled dTTP ((2′-deoxythymidine 5′-triphosphate)

Using 2,3,3-²H₃-serine (D3-serine) as a tracer, mitochondrial flux ismeasured by quantifying the labeling pattern of dTTP, a downstreamproduct of dTMP. Two-labeled (M+2) dTTP can only arise from cytosolicflux, since 1C units from the mitochondria enter the cytosol as formate,which can only retain one deuterium (FIG. 1A). Essentially all de novodTTP is M+1, implying nearly all 1C units originate in the mitochondria.In contrast, most de novo dTTP in ΔMTHFD2 cells (cells engineered with adeletion in MTHFD2) is M+2, meaning its 1C units derive from thecytosolic pathway. This assay is used to examine mitochondrial serineflux and the effect of compounds of the disclosure on mitochondrialserine flux, in either wildtype or mutant cells.

Methods: Isotope Labeling Experiments:

HEK-293T cells are cultured in 6-well plates in Dulbecco's modifiedeagle media (DMEM) without pyruvate with 10%/dialysed fetal bovine serumin 5% CO₂ at 37° C. Cells are treated with media containing uniformlylabeled ¹³C serine or 2,3,3-²H₃ serine (D3-serine) and, for inhibitorexperiments, either DMSO (vehicle) or compound of this disclosure (aninhibitor of SHMT2; racemic mixture or enatiomerically resolved). Growthis quenched and metabolites extracted by aspirating media, washing cellswith cold PBS, and immediately adding MeOH/water (80:20 at −80° C.).Supernatants from two rounds of extraction are combined, dried under N₂,resuspended in water, placed in a 4° C. autosampler, and analyzed byreverse-phase ion-paring chromatography negative-modeelectrospray-ionization high-resolution MS on a stand-alone orbitrap.

Deletion Cell Lines:

HEK293T cell lines (or other cell lines) mutant for SHMT1 and SHMT2 arecreated using the CRISPR-Cas9 system following standard publishedprotocols. Single clones from successful transfections are grown up,genotyped and characterized.

Example 6: Measurement of 5-Aminoimidazole-4-carboxamide ribonucleotide(AICAR)

Generation of AICAR provides an assay to evaluate one-carbon unitstress. Inhibition of SHMT2 in cells decreases serine metabolism andleads to one-carbon stress. Such cells show high constitutive levels of5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), a marker forone-carbon unit stress, when cultured in media. This can be rescued byexogenous treatment with sodium formate. Human cells expressing SHMT2are treated with control or increasing concentrations of an SHMTinhibitor, such as one in this disclosure.

Generation of AICAR provides an assay for evaluating compounds of thedisclosure. Compounds of the disclosure are evaluated in such an assay.

Example 7: Inhibition of Human SHMT

Inhibition of human SHMT by compounds in this disclosure is evaluated,for example, in their racemic forms in an in vitro assay. For example,Compound 61 (also referred to as KDG-30) was tested and demonstratedinhibition in vitro for both SHMT1 and SHMT2 with IC50 values at 3.2 nMand 0.78 nM respectively, shown in FIGS. 2A and 2B.

Of note, certain compounds in this disclosure are selective for SHMT.These compounds show activity against SHMT2 in vitro but do not inhibithuman MTHFD2 in enzymatic assays.

Enzymatic Assays:

For the SHMT1 and SHMT2 in vitro enzymatic assays, the rate of5,10-methylene tetrahydrofolate formation catalyzed by SHMT1/2 wasindirectly evaluated by coupling with excess MTHFD2, which converts NAD+to NADH allowing for reaction monitoring by absorption at 340 nm. Thereaction was started by addition of serine (1 mM final) to either humanSHMT1 or human SHMT2 (2 mcg/mL), and human MTHFD2 (25 mcg/mL) in abuffer of 50 mM potassium phosphate (pH 7.4), 0.3 mM tetrahydrofolate,7.5 mM dithiothreitol, 1.25 mM NAD+, and 4% DMSO. Inhibition of initialreaction velocity was determined by adding various inhibitors atdifferent concentrations and monitored as described. IC50 may becalculated based on this assay.

For the MTHFD2 in vitro assay, the rate of NADH formation is directlymonitored at 340 nm. The reaction is started by addition of 0.125 uM(final) of 5,10 methylene tetrahydrofolate to MTHFD2 (2.5 mcg/mL), 50 mMpotassium phosphate (pH 7.4), 7.5 mM dithiothreitol, 1.25 mM NAD⁺, and4% DMSO.

Example 8: Inhibition of Cancer Cell Growth

Inhibitors of SHMT were evaluated in various cancer cell lines, in thepresence and absence of mutations in mitochondrial folate pathwayenzymes. In this example, compound 61 (also referred to as KDG-30)inhibited growth (proliferation) of human colon carcinoma cells HCT116.The inhibitory activity of compound 61 was increased or potentiated inthe presence of a mutation in a mitochondrial folate pathway enzyme,specifically in SHMT2 knock-out HCT116 cells. As shown in FIG. 3A andFIG. 3B, compound 61 inhibited growth of both the native and knockoutHCT116 cancer cells, albeit with increased potency versus the SHMT2knock-out HCT116 cells.

Example 9: Inhibition of Human Cell Growth

Inhibitors of SHMT can be evaluated in cells in culture, such astransformed cells or cancerous cells, to evaluate inhibition of cellgrowth.

For example, Human Embryonic Kidney HEK293T cells (e.g., wild-typeHEK293T, SHMT2 knock-out HEK293T cells, and MTHFD2 knock-out HEK293Tcells) are cultured in 96-well plates in Dulbecco's modified eagle media(DMEM) without pyruvate with 10% dialysed fetal bovine serum(Invitrogen) in 5% CO₂ at 37° C. 24 hours after plating, cells aretreated with various concentrations of an inhibitor. Growth was seriallyassessed using the resazurin (‘AlamarBlue’) assay.

Example 10: Formate Rescue Assays

Application of formate is shown to rescue the anti-growth effect incertain human cells treated with SHMT inhibitors in a cell growth assay.

Cell Growth Assays

The described cell lines for Examples 8 and 9 are cultured in 96-wellplates in Dulbecco's modified eagle media (DMEM) without pyruvate(CELLGRO) with 10% dialysed fetal bovine serum (Invitrogen) in 5% CO₂ at37° C. Approximately 2,500 cells are plated in each well in 90 uL ofmedia. After 24 hours cells are treated with various concentrations ofinhibitor (time=0 hours), with or without formate. Growth was assessedat multiple time points (time=0, 24, 48, 72 hours) through measurementof fluorescence at 595 nm (excitation at 535 nm) on a BioTek micro titerplate reader after incubation with resazurin (0.01 mg/mL for 1.5 hours).IC50 curves are generated from percent inhibition of growth at 72 hours.

Example 11: SHMT Inhibitor Effective in Hematological Malignancies

Cell lines derived from neoplasias of hematological origin are sensitiveto the effects of SHMT inhibition. For example, growth (e.g., growth,proliferation and/or survival), as assessed by cell count, of both theT-cell leukemia line, Jurkat, and the diffuse large B cell lymphoma(DLBCL) line, Su-DHL-2, was inhibited by treatment with 5 μM of the SHMTinhibitor, HK-16 (PK2). For Jurkat cells, this inhibition issignificantly rescued by addition of 1 mM sodium formate to the growthmedia. In contrast, the DLBCL line Su-DHL-2 was not rescued by 1 mMformate. (FIGS. 4A and 4B).

Example 12: SHMT Inhibitor (SHMTi) Activity can be Rescued in Su-DHL-2Cells with Formate and Glycine

The inhibitory effects on growth of Su-DHL-2 cells was rescued bysupplementation with additional glycine (10× the standard RPMIconcentration, 100 mg/L total) and sodium formate (1 mM). A similarpattern was observed in a larger group of DLBCL lines (FIGS. 5 and 6).In all DLBCL lines tested, treatment with an SHMT inhibitor inhibitedcell growth (e.g., inhibited growth, proliferation and/or survival), andformate did not rescue this inhibitory effect. But, addition of bothformate and 10× glycine did rescue in approximately ¾ of the B celllines tested.

Example 13: Supplemental Formate and Glycine Required to Rescue B-CellLymphoma Lines from SHMTi Inhibition

Diffuse large B-cell lymphoma (DLBCL) lines (Su-DHL-2, Su-DHL-4, Farageand Su-DHL-6) were cultured in RPMI with 5 uM of an SHMTi, andinhibition of growth (e.g., inhibition of growth, proliferation and/orsurvival), as measured by cell count, was observed. This inhibition wasnot rescued by addition of formate to the culture media, but was largelyrescued by addition of both formate (1 mM sodium formate) AND glycine(100 mg/L). By comparison, in a representative AML cell line, such asREH, cell growth was rescued by addition of formate alone. Results areshown in FIG. 6.

Cellular Growth Assays:

Jurkat, REH, Farage, Daudi, Su-DHL2, Su-DHL-4, Su-DHL-6 cells werepurchased from ATCC. Cells were subcultured in RPMI supplemented with10% FBS. For growth assays, cells were seeded at 2×10{circumflex over( )}5/mL in 5 mL growth media in 12.5 cm flasks. Media was supplementedwith sodium formate (1 mM), additional glycine (100 mg/L) and an SHMTi(5 uM). Cells were sampled daily, and counted using trypan blue stainingand light microscopy.

Example 14: Glycine Uptake is Impaired in B-Cell Malignancies

1,2-13C Glycine Labeling Assays:

FIG. 7B shows the results of isotopic labeling (¹³C) of ADP extractedfrom various adherent solid tumor derived cancer cell lines incubatedwith 1,2-¹³C glycine for 48 hours as determined by mass spectrometry.Cell lines were cultured in standard culture media (RPMI for suspensionlines, DMEM for adherent lines), supplemented with 10% dialyzed FBS.Cells were grown in 1,2-¹³C glycine (replacing unlabeled glycine inmedia) containing media for 48 hr before harvesting for polarmetabolites. Suspension cells were pelleted, washed with PBS once andmetabolism quenched with −80 C lysis buffer (80:20 methanol:water).Metabolites were analyzed on a ThermoFisher Orbitrap mass spectrometeroperating in negative ion mode as previously described. Peak identitieswere confirmed using external standards and quantified using the MAVENsoftware suite6. In all adherent, solid tumor cell lines tested, asubstantial fraction of ADP is M+2 labeled, indicating incorporation of¹³C glycine from the media. In contrast, cell lines from both Burkittlymphoma and DLBCL showed nearly no incorporation of glycine into ADP(see M+2 results in FIG. 7C).

These data suggested that when compared to adherent cultured cancer celllines, B-cell derived cell lines do not import glycine from the mediainto biomolecules. The synthesis of the adenine ring in ADP involves 1glycine molecule and 2 carbons atoms from 2 equivalents of 10-formyl-THF(FIG. 7A). In adherent cells, replacement of glycine in standard cellculture media with labeled 1,2-¹³C glycine leads to generation of theM+2 labeling isomer of ADP. However, when the experiment was repeated inB-cell derived cancer cell lines (including Burkitt lymphoma (Daudi) andDLBCL), no labeling into ADP was observed from 1,2-¹³C glycine. When 10×glycine was used, labeling was observed confirming that the method ofrescue was glycine import into these cells (Data not shown).

Example 15: Requirement for SHMT Activity in HCT-116 Xenograft Formation

Clonal deletion cell lines of SHMT1, SHMT2, and SHMT1/2 were generatedfrom the human colorectal carcinoma cell line HCT-116. Paired Cas9nickase (Cas9n) containing constructs that encoded single-guide RNAsequences targeting SHMT1 or 2 were transiently transfected into cells,and mutant colonies from single clones were picked as described inDucker et al., (2016) Reversal of Cytosolic One-Carbon Flux Compensatesfor Loss of the Mitochondrial Folate Pathway, Cell Metab 23, 1140-1153.SHMT1 deletion had no effect on cell growth either in cell culture or assubcutaneous xenografts in nude mice. In contrast, SHMT2 deletion cellsgrew slower in culture and as xenografts (FIG. 8A). Liquidchromatography-mass spectrometry (LC-MS) analysis of the solublemetabolites extracted from SHMT2 deletion tumors revealed characteristicsigns of defective serine catabolism (FIG. 8B): serine levels wereincreased approximately 2-fold and the purine intermediateaminoimidazole carboxamide ribotide (AICAR), whose consumption requires10-formyl-THF, was elevated approximately 25-fold.

To generate dual SHMT1/SHMT2 double deletion cell lines, SHMT2 deletioncells were transfected with Cas9 and guide RNA sequences targeting SHMT1in the presence of 1 mM sodium formate. Isolated clones cultured informate grew at rates comparable to wild-type parental cells; aspredicted no growth was observed in media without formate (FIG. 8C). Totest whether circulating nucleotides and 1C sources in vivo couldsupport the growth of SHMT1/SHMT2 double deletion cells, we xenograftedthem into nude mice. No tumors were observed from the SHMT1/SHMT2 doubledeletion cells (FIG. 8D). Thus, in HCT-116 xenografts, circulatingalternative 1C donors (e.g. betaine, sarcosine, formate) and nucleotidesare together insufficient to support intracellular 1C metabolismrequired for tumorigenesis.

Example 16: Small Molecule Inhibitors of Human SHMT1/2

This disclosure provides compounds with improved inhibition propertiesfor human SHMT1 and/or SHMT2. These compounds were modestly more potentin vitro against SHMT1 than SHMT2. Changes that improve potency againstboth human isoforms include introduction of an isopropyl group at thechiral 4-carbon of the pyrano ring and adding steric bulk to the metasubstitutions on the phenyl ring (Compound HK-X1). Aromatic substitutionat this position further increased potency yielding Compound HK-X2,which inhibits T cell proliferation.

To understand the binding mode of these inhibitors, a 2.47 Å structureof human SHMT2 as a dimer in complex with glycine, PLP and racemic HK-X1(FIG. 9B) (PDB 5V7I). Electron density was identified in both bindingpockets of the protein dimer, but in only one active site was it wellresolved. Hydrogen binding contacts with the exocyclic amine are madewith the amide backbone of L166 and between the pyrazole and H171.Overlaying this inhibitor-bound structure with a previously solvedstructure of rabbit SHMT1 bound to 5-formyl-THF triglutamate (PDB 1LS3)revealed that the bicyclic ring system of HK-X1 and pteridine moiety offolate occupy the same space, but at a different angle (FIG. 9C). Thesubstituted phenyl ring and associated pyrrolidine of HK-X1 trace alongthe para-aminobenzoic acid (PABA) moiety of folate as it exits thepteridine binding pocket towards the solvent exposed folatepolyglutamate side chain. Directly adjacent to the pyrrolidine lies atyrosine residue that is well positioned to form a pi-stackinginteraction with the phenyl of HK-X2, potentially contributing to theimproved potency of this compound. Compared to the position of thefolate pteridine ring, the pyrazolopyran ring of HK-X1 is rotatedapproximately 60° out of plane. However, hydrogen bond contacts arepreserved, and engage the inhibitor at several core positions includingthe exocyclic amine and the pyrazole nitrogens. Given the conservednature of the SHMT active site, these compounds are likely to inhibitSHMT enzymes not only of humans, but also other mammals.

Both Compounds HK-X1 and HK-X2 contain a single chiral center. Althoughcrystallization was performed with racemic HK-X1, the electron densitywas consistent with only a single enantiomer binding to the enzyme.Using chiral chromatography, HK-X1 was separated, and enantioselectiveenzyme inhibition was confirmed (FIG. 9A). Thus, these pyrazolopyransare potent, folate-competitive, enantioselective mammalian SHMT1/2inhibitors.

Vibrational circular dichroism spectroscopy (VCD) will be used todetermine the absolute stereochemistry of the active enantiomer.However, it has been found that the active enantiomer of Compound HK-X1has a (+) optical rotation, and by structural analogy, it is expectedthat the active isomer of Compound HK-X2 will also have a (+) opticalrotation.

The activity of Compound HK-X1 and HK-X2 against cytosolic andmitochondrial SHMT isoforms in cultured cells was investigated. Theinactive (−) enantiomer of HK-X2 had no significant effect on growth inHCT-116 cells at doses up to 30 μM, whereas the active (+) enantiomerblocked growth with half-maximal inhibitory constants (ICs) of 870 nM(FIG. 9D, Table 5). To analyze the effects of inhibition on each isoformindependently, SHMT1 and SHMT2 HCT-116 deletion clones were used. Theactive enantiomers of both compounds, (+)-HK-X1 and (+)-HK-X2, werepotent against cytosolic SHMT1, as evidenced by IC₅₀s for growth of lessthan 50 nM in SHMT2 deletion cells (FIG. 9D, Table 5). In contrast,SHMT1 deletion cells showed indistinguishable sensitivity from wild-type(WT), confirming that mitochondrial SHMT inhibition is limiting forcompound efficacy (Table 5).

As HK-X1 and HK-X2 both have similar biochemical activities againstSHMT1 and SHMT2, the much higher doses required for functionalinhibition of cellular SHMT2 likely reflects a combination of imperfectmitochondrial penetration and greater intrinsic cellular SHMT2 activity(i.e., a substantial functional reserve due to high SHMT2 expression).Importantly, the effects on cell growth of HK-X1 and HK-X2 could berescued by addition of formate, indicating that they inhibit cell growththrough on-target depletion of cellular 1C pools (Table 5). However, asglycine is also a product of the SHMT reaction, formate can only rescuecell growth when this amino acid is present in the media.

TABLE 5 Cellular IC₅₀ (nM) Compound WT +formate ASHMT1 ASHMT2 (+)-HK-X12300 13500 2800 36 (+)-HK-X2 870 >50000 840 10

Notably, while most cancers have high mitochondrial 1C pathway activity,certain cancer cells, such as the pancreatic cancer cell line 8988T,harbor genetic lesions in the mitochondrial folate pathway activity andtherefore rely on SHMT1 to generate 1C units. In such cells, HK-X2impairs cell growth at concentrations <100 nM due to its potentengagement of cellular SHMT1 (FIG. 9E).

Example 17: Metabolic Readouts for Target Engagement

Inhibition of cellular SHMT activity can be monitored by isotope tracersand LC-MS. U-¹³C serine is catabolized in the mitochondria by SHMT2 intoU-¹³C-glycine and a ¹³C-5,10-methylene-THF (FIG. 10A). Glycine isfurther incorporated into downstream metabolites such as glutathione andpurines, whereas the folate 1C unit can be exported to the cytosol forincorporation into purines and thymidine. In addition, glycine and a 1Cunit can recombine to make partially labeled serine via SHMT1 or SHMT2.To assess target engagement, the effects of SHMT genetic manipulationsto pharmacological treatment with HK-X2 were compared. Serine mediaconsumption was inhibited in both HCT-116 SHMT1/2 double deletion cellsand WT cells treated with (+)-HK-X2 (FIG. 10B). Glycine production fromserine and subsequent incorporation into glutathione or ADP wascompletely blocked in SHMT1/2 double deletion cells as evidenced by themissing M+2 labeling fraction (FIG. 10C). Nearly complete blockade wasobserved in WT cells treated with (+)-HK-X2 but not (−)-HK-X2. Drugtreatment also blocked recombination of glycine and 10-formyl-THF toreform serine. Genetic deletion of SHMT1/2, and to a lesser extentSHMT2, results in buildup of purine biosynthetic intermediates upstreamof steps requiring 10-formyl-THF as a substrate (FIG. 10D). Such buildupis also seen with (+)-HK-X2. Thus, HK-X2 phenocopies, in anenantioselective manner, the metabolic consequences of SHMT geneticdeletion.

To assess the selectivity of the metabolic effects of HK-X2, untargetedLC-MS analysis on soluble metabolites from drug-treated cells wasperformed (FIG. 10F). In addition to purine intermediates, a buildup ofpurine salvage products (xanthosine, guanosine), whose increase isconsistent with purine insufficiency, was observed. Buildup ofhomocysteine, a classic marker of 1C deficiency was further observed.Depletion of the pyrimidine intermediate N-carbamoyl-aspartate was alsoobserved, likely reflecting feedback inhibition of aspartatetranscarbamoylase by excess pyrimidines in the purine starved cells.Importantly, there were no other large changes in metabolism suggestiveof off-target effects. Moreover, the changes in metabolite abundanceswere rescued by formate (FIG. 10F) and metabolite abundances in SHMT1/2double deletion cells closely matched those from WT cells treated with(+)-HK-X2. Thus, at doses sufficient to robustly inhibit SHMT1 and SHMT2in cell culture, (+)-HK-X2 selectively targets 1C metabolism.

Example 18: Cancer Cell Sensitivity to SHMT Inhibition

A panel of nearly 300 human cancer cell lines was screened for growth inthe presence of the (+)-enantiomer of HK-X1 (FIG. 11A). The median IC₅₀was 4 μM. Cell lines of B-cell lymphoma origin were enriched in the moresensitive half of cells (p<0.001, Fisher's Exact Test). This effect wasdriven by a pronounced sensitivity of Burkitt's and DLBCL lymphomas(FIG. 11A). A set of hematological cancer lines was then screened with(+)-HK-X2, supplemented with and without formate to test for rescue(FIG. 11B). Like HCT-116 cells, cell lines of T-cell origin such asAcute Lymphocytic Leukemia (ALL) cells, were largely rescued from theantigrowth effects of (+)-HK-X2 by formate. In contrast, formate failedto rescue the growth of B-cell lymphoma lines.

To explore this surprising lack of rescue further, flow cytometry wasused to analyze the effect of (+)-HK-X2, with and without formate, onthe DLBCL cell line Su-DHL-4. HK-X2 itself induced apoptosis as measuredby Annexin V surface staining (FIGS. 11C and 11D). Apoptosis wasenhanced by co-treatment with formate. In contrast, as expected, formaterescued Jurkat E6-1 leukemia cells from apoptosis (FIG. 11C). Withoutwishing to be bound by theory, it is hypothesized that the failure offormate to rescue growth in the DLBCL cell lines is due to a requirementfor both glycine and 1C units made by SHMT in these cells. When glycineis limiting, formate can enhance the cytotoxicity of SHMT inhibition.For example, formate augments the effect of HK-X2 in HCT-116 cells inglycine-free media. Mechanistically, by supplying 5,10-methylene-THF,formate may drives residual SHMT enzymatic function in theglycine-consuming direction. Alternatively, whereas cells may have themachinery to sense 1C deficiency and safely pause growth (e.g. due toAICAR activation of AMPK), they may lack comparable mechanisms forsurviving glycine limitation.

Example 19: Defective Glycine Uptake in DLBCL

The inability of formate to rescue the antigrowth effects ofenantiomerically pure HK-X2 ((+)-HK-X2) in DLBCL raised the question ofwhether HK-X2 activity in these cells was on target. If so, it washypothesized that the cells might require SHMT not only for 1C units,but also to produce glycine. Indeed, SHMT2 loss causes glycineauxotrophy. Here, however, the cells were cultured in RPMI, whichcontains glycine (10 mg/L, 130 μM). Accordingly, the goal of thisexperiment is to understand if DLBCL and other B-cell lines might bedeficient in glycine uptake. Compared to adherent cancer cell lines, andhematological cancer lines that were rescued by formate, B-cell cancerlines uptake glycine and incorporate it into downstream metabolites suchas purines and glutathione at lower rates (FIG. 12A).

The defect in glycine uptake was particularly profound in certain DLBCLlines, such as Su-DHL4 which is sensitive to HK-X2 and not rescued byformate (FIG. 12B). Jurkat cells are actually more sensitive to HK-X2than SuDHL4 cells, but their growth can largely be rescued by formate(FIG. 12C). In contrast, cell growth is further decreased in Su-DHL4cells by addition of formate. This can be further exacerbated by removalof glycine from the media. The toxic effects of formate in the presenceof HK-X2 are likely due to the resulting methylene-THF shifting thethermodynamics of the SHMT reaction in the direction of glycineconsumption. Using flow cytometry, apoptosis of Su-DHL4 cells werestudied with AnnexinV surface staining (FIGS. 12D and 12E). Withinhibitor alone, the apoptotic fraction of cells was increased from 2%to 8% and further doubled in the presence of formate to 15%.

To confirm the hypothesis that glycine availability was limiting inSu-DHL4 cells and to explain the synergistic cell killing effects ofHK-X2 in combination with formate, how varying RPMI glycineconcentration would alter the cell response to the SHMT inhibitor, e.g.,HK-X2, was studied. In SuDHL4 cells, the IC₅₀ of HK-X2 was highlydependent upon the glycine concentration. Increasing or decreasingglycine by 10-fold compared to standard RPMI markedly shifted the HK-X2IC₅₀ values (FIG. 12F). For Jurkat cells, little dependence uponextracellular glycine concentrations was observed. Across a set of DLBCLcell lines, supplying both formate and supra-physiologic glycinegenerally rescued cell growth, indicating on-target HK-X2 activity withboth products of the SHMT reaction being required (FIG. 12G).

HK-X2 induced glycine deficiency in DLBCL cells, even though they werecultured in complete media with glycine (RPMI, 10 mg/L glycine=130 μM).This suggested that glycine uptake is intrinsically impaired in thesecells. Using U-¹³C-glycine, the kinetics of extracellular glycineincorporation into cells and downstream metabolic products was monitored(FIG. 14D). Labeling of intracellular glycine products, such asglutathione and ADP, was markedly less in Su-DHL-4 cells than Jurkatcells. In a larger set of cell lines, composed of both otherhematological cancer and adherent cell lines, steady state labeling ofintracellular metabolites from glycine was significantly lower in B-celllymphoma cell lines (FIG. 14E).

Given the apparent glycine shortage in these B cells upon HK-X2treatment, extracellular glycine levels were augmented, and response todrug was evaluated. First, the concentration of glycine in RPMI wasaltered and response to drug observed. A reduction of glycine in themedia modestly improved the potency of HK-X2, indicating that the cellswere sensitive to extracellular glycine. More strikingly, increasing themedia glycine by 10-fold substantially rescued the cells from HK-X2. Incontrast, in Jurkat cells, a small amount of extracellular glycine wassufficient and more did not further rescue the cells from HK-X2. Acrossa set of DLBCL cell lines, representing both ABC and GBC subtypes,supplying both formate and supra-physiologic glycine (100 mg/L, 1.3 mM)generally rescued cell growth (FIG. 12G). These results indicate theimportance of both products of the SHMT reaction, glycine and folate 1Cunits, for the proliferation of DLBCL cell lines.

Knowing that manipulating glycine could augment the efficacy of HK-X2,different mechanisms of decreasing glycine were tested. When formate wasadded, HK-X2 was transformed from being a drug that slowed cell growthto one that was fully cytostatic (FIG. 14F). Further removing glycinecaused significant cell death. Interestingly, combining the glycinereuptake transporter 1 (GlyT) (SLC6A9) inhibitor RG1678 with HK-X2 ledto even greater cell death, even in the presence of media glycine (FIG.14F). These results suggest that the low level of glycine uptake inthese cells is mediated by GlyT and that combinations of formate, SHMTinhibitor, and/or GlyT inhibitor may selectively target these cells.

Example 20: DLBCL Cells Require SHMT to Make Glycine for PurineSynthesis

The inability of formate to rescue the antigrowth effects of HK-X2 inDLBCL cell lines suggested that glycine may be limiting in these cells.To explore this hypothesis, the metabolic effects of HK-X2 in DLBCL andJurkat cells treated with (+)-HK-X2 (72 h, 5 μM) with and withoutformate were characterized. In Jurkat and DLBCL cell lines Su-DHL-4 andSu-DHL-2, HK-X2 treatment led to a large reduction in nucleotidetriphosphates (FIG. 14A). This can be rationalized as reflectingimpaired purine synthesis, which requires both 1C units and glycine,with pyrimidines also falling due to endogenous mechanisms that balancetheir levels with those of purines. There is also a component of energystress, particularly in Su-DHL-4 cells, as nucleotide monophosphateswere increased, not decreased. Consistent with 1C limitation, dTTP,whose synthesis requires a folate 1C unit, was more depleted than otherpyrimidines.

Formate supplementation restored nucleotide levels in Jurkat but notDLBCL cell lines. It was confirmed that formate rescues folate 1C levelsin DLBCL cells, as the AICAR accumulation induced by (+)-HK-X2 is fullyreversed. Thus, while nucleotide synthesis in HK-X2-treated Jurkat cellsis solely limited by 1C units, an additional factor is lacking in DLBCLcells. Consistent with glycine being the second factor missing in DLBCLcells, (+)-HK-X2 treatment depleted the glycine-containing redox defensetripeptide glutathione (FIG. 14B). Strikingly, while HK-X2 alone did notalter glutathione in Jurkat cells, formate addition caused glutathionedepletion. This further validates that, when SHMT is inhibited,provision of excess 1C units can cause glycine stress. Glutathionesupplementation did not rescue growth. Based on these results, it washypothesized that growth in SHMT-inhibited DLBCL cells might be restoredwith purine supplementation which would simultaneously alleviate 1C andglycine metabolic stress. Growth was partially rescued in Su-DHL-4 cellstreated with hypoxanthine. Thymidine, which rescues the effects of theclassical antifolate pemetrexed but does not contain glycine, had nobenefit in HK-X2-treated DLBCL cells (FIG. 14C). Thus, HK-X2 blocks cellgrowth through a progressive depletion of purines leading to loss ofnucleotide triphosphates. Restoration of purines levels restores growth.

Whether glycine shortage might also impact protein synthesis was nextexamined. Severe amino acid shortages lead to loss of cognate tRNAcharging and thus ribosome stalling, which can be measured usingribosome profiling. Ribosome profiling on Su-DHL-4 cells treated with(+)-HK-X2 was performed. Untreated Su-DHL-4 cells growing in RPMI didnot show evidence of glycyl-tRNA insufficiency; no enrichment for thesecodons was observed. Furthermore, no difference in glycine codonoccupancy was observed between treated and control cells. Collectively,these results suggest a hierarchy in the sensitivity of differentintracellular metabolic products to glycine levels: glutathionesynthesis is most sensitive, followed by purine synthesis, with proteinsynthesis most resistant. This hierarchy is consistent with biochemicalmeasurements of the K_(M) values of the relevant enzymes: theglycyl-tRNA amino acid synthase has a lower K_(M) for glycine (15 μM)than that found in glycinamide ribonucleotide synthetase (45 μM) orglutathione synthetase (452 μM).

Example 21: Liver Microsome Assay

Male CD-1 mouse liver microsomes were obtained from commercial sources(Corning #452701). The following master solution was prepared beforecompound addition:

Stock Final Reagent Concentration Volume Concentration Phosphate buffer200 mM 200 μL  100 mM Ultra-pure H₂O —  64 μL — MgCl₂ solution  50 mM 40 μL   5 mM Alamethacin  5 mg/mL  2 μL   25 ug/mL Microsomes  20 mg/mL 10 μL  0.5 mg/mL

Two separate experiments were performed as follows: a) With Cofactors(NADPH and UDPGA): 40 μL of 10 mM NADPH and 40 μL of 20 mM UDPGA wereadded to the incubations. The final concentrations of microsomes, NADPHand UDPGA were 0.5 mg/mL, 1 mM and 2 mM, respectively. b) WithoutCofactors (NADPH and UDPGA): 10 μL of 20 mg/mL liver microsomes and 80μL of ultra-pure water were added to the incubations. The finalconcentration of microsomes was 0.5 mg/mL. The mixture was pre-warmed at37° C. for 5 minutes.

The reaction was started with the addition of 4 μL of 200 μM controlcompound or test compound solution. Verapamil was used as positivecontrol in this study. The final concentration of test compound orcontrol compound was 2 μM. The incubation solution was incubated inwater bath at 37° C. Aliquots of 50 μL were taken from the reactionsolution at 0.5, 5, 15, 30, 45 and 60 minutes. The reaction was stoppedby the addition of 5 volumes of cold acetonitrile with IS (100 nMalprazolam, 200 nM caffeine and 100 nM tolbutamide). Samples werecentrifuged at 3,220 g for 40 minutes. Aliquot of 100 μL of thesupernatant was mixed with 100 μL of ultra-pure H₂O and then used forLC-MS/MS analysis. The in vitro half-life (in vitro t1/2) was determinedfrom the slope value of the LC-MS signal of the test compound. The dataare reported in Table 6.

TABLE 6 Metabolic Stability of Test Compounds in Pooled Male Mouse LiverMicrosomes CL_(int) CL_(int) t1/2 (μL/min/mg t1/2 (μL/min/mg Replicateprotein) Replicate protein) Compound No./Structure 1 (replicate 1) 2(replicate 2)  1 2.32 596.63 1.79 776.05 71 4.86 285.28 6.84 202.99 111.82 763.82 2.56 541.48 74 20.40 67.95 20.40 67.96 HK-X3 15.09 91.8714.70 94.28

15.79 87.77 19.39 71.47

Other SHMT inhibitors, such as compounds of any of Formulae (I)-(IX),are evaluated in similar assays, such as the assays set forth inExamples 10-19.

All publications and patents cited herein are hereby incorporated byreference in their entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the subject matters in this disclosure described herein.Such equivalents are intended to be encompassed by the following claims.

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The invention claimed is:
 1. A method for inhibiting a serinehydroxymethyltransferase (SHMT), comprising contacting the SHMT with acompound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R⁰, R¹ and R²are each independently selected from the group consisting of —H,halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹²,—OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted haloalkyl, and substituted or unsubstituted haloalkoxy;provided that at least one of R⁰, R¹ and R² is selected from the groupconsisting of substituted or unsubstituted alkenyl, and substituted orunsubstituted alkynyl; R³ is selected from the group consisting of —H,halogen, hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹²,—OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted haloalkyl, and substituted or unsubstituted haloalkoxy; R⁴is selected from the group consisting of —H, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, and substituted orunsubstituted heteroarylalkyl; R⁵, R⁶ and R⁷ are each independentlyselected from the group consisting of —H, —C(O)R¹¹, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, and substituted orunsubstituted heteroarylalkyl; or R⁵ is selected from any of theforegoing and R⁶ and R⁷ taken together with the nitrogen atom to whichthey are attached form a substituted or unsubstituted 3-6 membered ring;each occurrence of R¹¹ is independently selected from the groupconsisting of substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl; and each occurrence of R¹⁰ and R¹² is independently selectedfrom the group consisting of —H, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.
 2. The method according to claim 1, whereinthe compound is a compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein: W represents—CR¹⁶═CR¹⁶— or —C≡C—; n is 0, 1, 2, 3, or 4; R¹³, R¹⁴, and R¹⁵ areindependently selected from the group consisting of hydrogen, —OH,halogen, optionally substituted alkyl, optionally substituted haloalkyl,—OR^(a), —OC(O)R^(b), —C(O)NR^(a)R^(b), and —NR^(a)R^(b); or R¹³ and R¹⁴together with the atom to which they are attached form a 4-7 memberedheterocyclic ring comprising 1 or 2 heteroatoms selected from the groupconsisting of NR^(a), O, S, SO, or SO₂; wherein the heterocyclic ring isoptionally substituted with one or more substituents independentlyselected from the group consisting of oxo and optionally substitutedalkyl; and R¹⁶, R^(a) and R^(b), independently at each occurrence, are Hor optionally substituted alkyl.
 3. The method according to claim 2,wherein W is —C≡C—.
 4. The method according to claim 2, wherein R¹³,R¹⁴, and R¹⁵ are independently selected from the group consisting ofhydrogen, —OH, halogen, optionally substituted alkyl, optionallysubstituted haloalkyl, —OR^(a), —OC(O)R^(b), —C(O)NR^(a)R^(b), and—NR^(a)R^(b), wherein R^(a) and R^(b), independently at each occurrence,are H or optionally substituted alkyl.
 5. The method according to claim1, wherein the compound is a compound of formula (III):

or a pharmaceutically acceptable salt thereof, wherein: W represents—CR¹⁶═CR¹⁶— or —C≡C—; R¹⁶ is H or optionally substituted alkyl; and Arepresents optionally substituted aryl or optionally substitutedheteroaryl.
 6. The method according to claim 5, wherein W is —C≡C—. 7.The method according to claim 5, wherein A is aryl, optionallysubstituted with one or more substituents independently selected fromthe group consisting of —OH, halogen, optionally substituted alkyl,optionally substituted haloalkyl, —OR^(a), —OC(O)R^(b),—C(O)NR^(a)R^(b), and —NR^(a)R^(b), wherein R^(a) and R^(b),independently at each occurrence, are H or optionally substituted alkyl.8. The method according to claim 7, wherein A is phenyl, optionallysubstituted with one or more substituents independently selected fromthe group consisting of —CH₂OH, —OH, —CF₃, —COOH, —F, —CH₂NH₂, —CONH₂,and —NH₂.
 9. The method according to claim 5, wherein A is heteroaryl,optionally substituted with one or more substituents independentlyselected from the group consisting of —OH, halogen, optionallysubstituted alkyl, optionally substituted haloalkyl, —OR^(a),—OC(O)R^(b), —C(O)NR^(a)R^(b), and —NR^(a)R^(b), wherein R^(a) andR^(b), independently at each occurrence, are H or optionally substitutedalkyl.
 10. The method according to claim 9, wherein A is an optionallysubstituted heteroaryl containing 1-4 N atoms.
 11. The method accordingto claim 2, wherein: R² is nitro, —F, —Cl, —OCH₃, —CCl₃, or —CF₃; R³ isselected from the group consisting of isopropyl, cyclopropyl, andcyclobutyl; R⁴ is methyl or isopropyl; and R⁵, R⁶ and R⁷ are eachindependently selected from the group consisting of —H, alkyl, phenyl,and —COCH₃.
 12. The method according to claim 1, wherein: R⁰ is —H; oneof R¹ and R² is substituted or unsubstituted alkenyl, or substituted orunsubstituted alkynyl; the other is independently selected from thegroup consisting of —H, halogen, hydroxyl, nitro, nitrile, —OR¹¹,substituted or unsubstituted alkyl, substituted or unsubstitutedalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted haloalkyl, or substituted or unsubstituted haloalkoxy; R³is selected from the group consisting of isopropyl, cyclopropyl, andcyclobutyl; R⁴ is methyl or isopropyl; and R⁵, R⁶ and R⁷ are eachindependently selected from the group consisting of —H, alkyl, phenyl,and —COCH₃.
 13. The method according to claim 1, wherein: R⁰ is —H; oneof R¹ and R² is substituted or unsubstituted alkenyl or substituted orunsubstituted alkynyl; the other is independently selected from thegroup consisting of —H, methoxy, fluoro, chloro, bromo, hydroxyl, nitro,nitrile, substituted or unsubstituted alkyl, —CCl₃, and —CF₃; R³ iscyclobutyl or iso-propyl; R⁴ is methyl; R⁵ and R⁶ are each independentlyselected from the group consisting of —H, alkyl, and phenyl; and R⁷ is—H.
 14. The method of claim 1, wherein the compound is represented bythe following structural formula:

or a pharmaceutically acceptable salt thereof.
 15. The method of claim1, wherein the SHMT is SHMT2.
 16. The method of claim 1, wherein theSHMT is SHMT1.
 17. The method of claim 1, comprising contacting the SHMTwith the compound, or a pharmaceutically acceptable salt thereof, invitro.
 18. The method of claim 1, comprising contacting the SHMT withthe compound, or a pharmaceutically acceptable salt thereof, in vivo.19. A method for inhibiting serine flux or folate metabolism in a cell,comprising contacting the cell with a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R⁰, R¹ and R²are each independently selected from the group consisting of —H,halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹²,—OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted haloalkyl, and substituted or unsubstituted haloalkoxy;provided that at least one of R⁰, R¹ and R² is selected from the groupconsisting of substituted or unsubstituted alkenyl, and substituted orunsubstituted alkynyl; R³ is selected from the group consisting of —H,halogen, hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹²,—OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted haloalkyl, and substituted or unsubstituted haloalkoxy; R⁴is selected from the group consisting of —H, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, and substituted orunsubstituted heteroarylalkyl; R⁵, R⁶ and R⁷ are each independentlyselected from the group consisting of —H, —C(O)R¹¹, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, and substituted orunsubstituted heteroarylalkyl; or R⁵ is selected from any of theforegoing and R⁶ and R⁷ taken together with the nitrogen atom to whichthey are attached form a substituted or unsubstituted 3-6 membered ring;each occurrence of R¹¹ is independently selected from the groupconsisting of substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl; and each occurrence of R¹⁰ and R¹² is independently selectedfrom the group consisting of —H, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.
 20. A method for inhibiting glycine generationin mitochondria or cytosol of a cell, comprising contacting the cellwith a compound of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein: R⁰, R¹ and R²are each independently selected from the group consisting of —H,halogen, hydroxyl, nitro, nitrile, —SOR¹¹,—S(O)₂R¹¹, —S(O)₂NR¹⁰R¹²,—OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocyclyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedarylalkyl, substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted haloalkyl, and substituted or unsubstituted haloalkoxy;provided that at least one of R⁰, R¹ and R² is selected from the groupconsisting of substituted or unsubstituted alkenyl, and substituted orunsubstituted alkynyl; R³ is selected from the group consisting of —H,halogen, hydroxyl, nitro, nitrile, —SOR¹¹, —S(O)₂R¹¹, —S(O)₂NR¹⁰R¹²,—OR¹¹, —OC(O)R¹², —C(O)OR¹², —C(O)R¹¹, —C(O)NR¹⁰R¹², —NR¹⁰R¹²,—N(R¹²)C(O)R¹¹, —NS(O)₂R¹², substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted arylalkyl,substituted or unsubstituted heteroarylalkyl, substituted orunsubstituted haloalkyl, and substituted or unsubstituted haloalkoxy; R⁴is selected from the group consisting of —H, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, and substituted orunsubstituted heteroarylalkyl; R⁵, R⁶ and R⁷ are each independentlyselected from the group consisting of —H, —C(O)R¹¹, substituted orunsubstituted alkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted arylalkyl, and substituted orunsubstituted heteroarylalkyl; or R⁵ is selected from any of theforegoing and R⁶ and R⁷ taken together with the nitrogen atom to whichthey are attached form a substituted or unsubstituted 3-6 membered ring;each occurrence of R¹¹ is independently selected from the groupconsisting of substituted or unsubstituted alkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl; and each occurrence of R¹⁰ and R¹² is independently selectedfrom the group consisting of —H, substituted or unsubstituted alkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocyclyl, substituted or unsubstituted aryl, and substituted orunsubstituted heteroaryl.